Treatment of neurodegenerative diseases

ABSTRACT

Use of selenate or a pharmaceutically acceptable salt thereof in methods and compositions for enhancing the activity of the protein phosphatase PP2A is provided. Methods of reducing phosphorylation of tau protein, inhibiting activity of GSK3 and treating or preventing neurodegenerative diseases are also described.

FIELD OF THE INVENTION

This invention relates to the use of selenate or a pharmaceuticallyacceptable salt thereof in methods and compositions for enhancing theactivity of PP2A. The present invention also relates to the use ofselenate or a pharmaceutically acceptable salt thereof in methods ofinhibiting or reducing phosphorylation of tau protein, in methods ofinhibiting the activity of GSK3β and particularly in methods of treatingor preventing neurodegenerative diseases. In some embodiments, theinvention relates to the use of selenate or a pharmaceuticallyacceptable salt thereof in combination with other therapies for use inmethods of treating or preventing neurodegenerative diseases.

BACKGROUND OF THE INVENTION

Neurodegenerative disease is a general term for a number of disordersthat act by compromising the brain's capacity to control itself or thebody by damaging neurons that facilitate normal brain function.Neurodegenerative diseases are primarily diseases of older people. Withan increase in life expectancy, the world's population is living longerand the number of sufferers of neurodegenerative diseases has beenincreasing.

In a number of neurodegenerative disorders, there is a deposition ofabnormal tau protein in neurons and glial cells in the brain. Forexample, abnormal tau protein has been found in neurofibrillary tanglescharacteristic of Alzheimer's disease (AD). Neurofibrillary tangles(NTs) are one of the two neuropathological hallmarks of AD [Lee et al.,2001]. Paired helical filaments (PHFs) are the major structuralcomponent of NTs and are mainly composed of microtubule-associatedprotein tau [Lee et al., 1991, Grundke-Iqbal et al. 1986a, Grundke-Iqbalet al., 1986b]. PHF-tau (tau protein isolated from PHFs) is highlyinsoluble, displays retarded mobility on SDS gel and is incapable ofbinding to microtubules because it is abnormally phosphorylated(phosphorylated on more sites than in normal tau protein) [Lee et al.,1991, Grundke-Iqbal et al. 1986a, Grundke-Iqbal et al., 1986b]. Upondephosphorylation, PHF-tau becomes soluble and as capable as normal tauprotein in binding and promoting microtubule assembly [Wang et al.,1995, Wang et al., 1996, Bramblett et al., 1993]. Abnormal tauphosphorylation is believed to cause tau dysfunction, microtubuleinstability, axonal transport loss, neurodegeneration and dementiaassociated with AD [Alonso et al., 1996].

One type of abnormal tau protein is hyperphosphorylated tau protein. Tauprotein is known to be phosphorylated at a number of phosphorylationsites by glycogen synthase kinase 3β (GSK3β) in vivo, including theAlzheimer's disease specific Ser³⁹⁶ residue [Li and Paudel, 2006]. Inturn, GSK3β is known to be phosphorylated by the protein kinase Akt andthe activity of Akt is known to be attenuated by the protein phosphatasePP2A.

PP2A is a heterotrimeric holoenzyme that exists in multiple formscomposed of a common core structure bound to different regulatorysubunits [Mumby and Walter, 1993]. The core enzyme is a complex betweenthe catalytic (C) and structural (A) subunits. A third class of subunit,termed B, comprises several polypeptides that regulate PP2A activity andspecificity [Mumby and Walter, 1993, Kamibayashi et al. 1994]. Asignificant portion of the ABC isoform of PP2A is associated withneuronal microtubules [Sontag et al., 1995], implicating PP2A in theregulation of the phosphorylation state of microtubule-associatedproteins (MAPs), such as tau. PP2A containing the B regulatory subunits,but not other forms of PP2A, (i.e. B′ and B″) has been shown to bind andpotently de-phosphorylate tau in vitro. Furthermore, inhibiting the ABCisoform of PP2A induces hyperphosphorylation of tau, dissociation of taufrom microtubules and loss of tau-induced microtubule stabilisation[Sontag et al., 1996]. It has recently been shown that PP2A accounts forapproximately 71% of the total tau phosphatase activity of human brain[Liu et al., 2005]. The total phosphatase activity and the activities ofPP2A toward tau are significantly decreased in brains of AD patientswhereas that of other phosphatases such as PP2B are actually increasedin the AD brain [Liu et al., 2005]. PP2A activity negatively correlatesto the level of tau phosphorylation at most phosphorylation sites inhuman brains. This indicates that PP2A is the major tau phosphatase thatregulates its phosphorylation at multiple sites in human brain. Thisimplies that the abnormal hyperphosphorylation of tau is partially dueto a downregulation of PP2A activity in AD brain and that agents thatcan act to boost the activity of PP2A and in particular the ABC isoformof PP2A would have clinical utility in treating and or preventingdevelopment of neurodegenerative diseases.

There is also increasing evidence that abnormal phosphorylation of tauprotein may be associated with neurodegenerative disorders in whichabnormal α-synuclein protein is present. Tau and α-synuclein pathologyboth occur in Alzheimer's disease, Parkinson's disease,Guam-Parkinson-ALS-dementia complex and Parkinson's disease caused bymutations in α-synuclein (Duda et al., 2002, Forman et al., 2002,Ishizawa et al., 2003).

The protein α-synuclein appears to play an important role in thepathophysiology of Parkinson's disease (PD). Lewy bodies are apathological hallmark of PD that are composed primarily of α-synuclein(Spillantini et al., 1997; Spillantini et al., 1998b).

α-Synuclein is thought to play a critical role in the pathophysiology ofPD because it accumulates in Lewy bodies and because genetic studiesidentified mutations in α-synuclein that are associated with familial PD(Kruger et al., 1998; Polymeropoulos et al., 1997; Singleton et al.,2003; Spillantini et al., 1998b; Zarranz et al., 2004).

The A53T and A30P mutations in α-synuclein appear to be causative in PDby increasing the tendency of α-synuclein to aggregate. The mutationsboth increase the tendency of α-synuclein to aggregate spontaneously orin response to exogenous factors, such as metals and oxidative stress(Conway et al., 2000; Hashimoto et al., 1999; Kruger et al., 1998;Ostrerova-Golts et al., 2000; Paik et al., 1999, 2000; Polymeropoulos etal., 1997).

The A53T and A30P mutations in α-synuclein also cause age-dependentα-synuclein aggregation and neuronal injury in transgenic mice andDrosophila (Feany and Bender, 2000; Giasson et al., 2002; Kahle et al.,2001; Masliah et al., 2000). These results emphasize the relevance ofα-synuclein to the study of neurodegeneration.

Abnormal phosphorylated tau is present in Lewy bodies found in sporadicPD patients and occurs in neurons near areas containing α-synucleinpathology (Ishizawa et al., 2003). In vitro evidence also linksα-synuclein and tau as α-synuclein binds tau in vitro, and stimulatestau phosphorylation by protein kinase A in vitro (Giasson et al., 2003;Jensen et al., 1999). Recent results indicate that α-synuclein enhancestau fibrillization in vitro and that abnormal tau fibrils are present inthe brains of symptomatic transgenic mice overexpressing mutant A53Tα-synuclein (Giasson et al., 2003).

Frasier M et al. 2005 have shown that A30P α-synuclein aggregationoccurs alongside tau pathology and that α-synuclein aggregation occursin parallel with tau pathology in transgenic mice overexpressing A30Pα-synuclein, and have shown that symptomatic A30P α-synuclein transgenicmice exhibit abnormal tau phosphorylation and that the phosphorylationcorrelates with activation of a c-jun kinase.

Apart from aggregation of α-synuclein (α-Syn), oxidative stress andexposure to certain neurotoxins such as1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are linked to thepathogenesis of PD. MPTP induces a selective degeneration of thenigrostriatal dopaminergic pathway in mice and primates, as seen in PD,associated with increases in α-Syn expression levels and aggregation,but without inducing real Lewy bodies (Dauer and Przedborski, 2003), buttriggering formation of nigral inclusions immunoreactive for ubiquitinand α-synuclein on continuous administration of MPTP (Fornai et al.,2005). Another component of certain Lewy bodies is abnormallyphosphorylated tau (Ishizawa et al., 2003). Neuronal colocalization oftau and α-Syn as aggregates or inclusions, or inside certain Lewy bodiesor Lewy body-like inclusions, has been reported in brains of patientswith familial Alzheimer's disease (AD), Down's syndrome, and Lewy bodydisease (Kotzbauer et al., 2001; Lippa et al., 1999; Arima et al., 1999;Iseki et al., 1999).

Similarities between tau and α-Syn include expression in presynapticneurons, long half-lives in vivo, their “natively unfolded” natureallowing for their heat stability, and their propensity to fibrillizethrough stretches of hydrophobic residues that form the core ofassembled fibrils (Friedhoff et al., 2000; Serpell et al., 2000).

Other in vitro data with the [396/404]S 3E double mutant (apseudophosphorylation construct mimicking the PHF-1 phosphorylationsites in which the two serine residues at position 396 and 404 in thelongest human isoform of tau, ht40, were replaced into glutamateresidues) tau also indicate that hyperphosphorylation at Ser396/404 maycause the C terminus of tau to assume a more extended conformation,altering its inhibitory effect on tau oligomerization and potentiatingthe rate of filament formation (Abraha et al., 2000).

Duka T et al., 2006, have now shown that MPTP-induced increases in α-Synexpression levels in mesencephalic dopaminergic neurons promote changesin the phosphorylation patterns of tau at the PHF-1 binding site(Ser396/404), resulting in a mislocation of both proteins and withincreased coimmunoprecipitation, together with increased levels ofsarkosyl-insoluble hyperphosphorylated tau, suggesting that an initialstep in MPTP-induced parkinsonism and neurotoxicity, is α-Syn-directedhyperphosphorylaton of Tau at Ser396/404.

The findings that MPTP causes increased dissociation of α-Syn frommicrotubules, together with decreases in phosphorylated-tau levelsassociated with the cytoskeletal bound fraction, may also be ofrelevance to the mechanism(s) underlying the neurodegenerative process.Hyperphosphorylation of tau greatly reduces the affinity of tau formicrotubules, causing their destabilization (Drechsel et al., 1992;Biernat et al., 1993; Michaelis et al., 2002). In addition,phosphorylated-tau is also known to bind to and deplete othermicrotubule binding proteins, such as MAP1 and MAP2, from microtubules(Iqbal and Grundke-Iqbal, 2005). Moreover, since α-Syn is known to bindto microtubules (Wersinger and Sidhu, 2005) with a possible role inaxonal transport (Sidhu et al., 2004), it is likely that thedissociation of this protein from microtubules further aggravates theinstability of microtubules, disrupting the cytoskeletal network andcellular homeostasis. Thus, the dissociation of α-Syn from microtubulesand abnormalities in the properties of tau bound to microtubules, maycomprise another link in the chain of events leading to theneurodegenerative processes associated with inclusion formation.

MPTP-induced abnormalities in α-Syn levels modulate phosphorylated-tauformation, and the PHF-1 form of tau, in particular, provide insights inthe development of the early phases of both PHF formation and associatedloss of vital neuronal function and suggest that MPTP-inducedparkinsonian syndromes or neurotoxicity may be a tauopathy withconcomitant alterations in α-Syn in a manner reminiscent ofsynucleopathies.

This suggests that abnormalities of a protein (tau) known to bemobilized during the pathogenesis of AD, may also be mobilized inparkinsonism but in a region of the brain not associated with AD,thereby suggesting considerable overlap in the genesis of certainneurodegenerative diseases. This suggests that neurodegenerativediseases that seem unrelated may actually have common triggering eventsand subsequent pathologies, which sets in motion neuronal degeneration.

There is a need for agents that affect the phosphorylation of tauprotein and are clinically useful in the treatment or prevention ofneurodegenerative disorders.

SUMMARY OF THE INVENTION

The present invention is predicated in part on the discovery that theactivity of the protein phosphatase PP2A can be enhanced by exposure toselenate or a pharmaceutically acceptable salt thereof. The enhancementof the activity of PP2A can reduce or inhibit phosphorylation of tauprotein, especially hyperphosphorylation, with a two pronged approach:i) dephosphorylation and inactivation of Akt, thereby reducingphosphorylation of GSK3β and consequently reducing phosphorylation oftau protein, and ii) direct dephosphorylation of tau protein. Areduction in the phosphorylation, especially hyperphosphorylation of tauprotein reduces or prevents the accumulation or deposition of abnormaltau protein in neurons and glial cells and therefore is useful in thetreatment or prevention of neurodegenerative disorders.

Accordingly, in one aspect, the present invention provides a method forthe treatment or prevention of a neurodegenerative disease in a subjectcomprising administering to the subject an effective amount of selenateor a pharmaceutically acceptable salt thereof. In some embodiments theneurodegenerative disease is a tauopathy. In some embodiments theneurodegenerative disease is an α-synucleopathy. In specificembodiments, the neurodegenerative disease is selected from preseniledementia, senile dementia, Alzheimer's disease and Parkinson's disease.

In another aspect of the invention there is provided a method ofinhibiting or reducing phosphorylation of a tau protein in a neuron,glial cell or Lewy body, comprising exposing the neuron or glial cell toan effective amount of selenate or a pharmaceutically acceptable saltthereof. In some embodiments, the tau protein is amicrotubule-associated tau protein. In some embodiments, the tau proteinis in a neurofibrillary tangle. In some embodiments,hyperphosphorylation of tau protein is inhibited or prevented.

In yet another aspect, the present invention provides a method ofenhancing the activity of PP2A comprising exposing the PP2A to aneffective amount of selenate or a pharmaceutically acceptable saltthereof. In some embodiments, the PP2A is an isoform thatdephosphorylates Akt. In some embodiments, the PP2A is an isoform thatdephosphorylates tau proteins, especially microtubule-associated tauproteins found in neurons and glial cells and Lewy bodies.

In a further aspect, the present invention provides a method ofinhibiting the activity of GSK3β in a neuron or glial cell, comprisingexposing the neuron or glial cell to an effective amount of selenate ora pharmaceutically acceptable salt thereof.

In a further aspect of the invention, there is provided a use ofselenate or a pharmaceutically acceptable salt thereof in themanufacture of a medicament for treating or preventing aneurodegenerative disease.

In some embodiments of the methods and uses broadly described above, theselenate or a pharmaceutically acceptable salt thereof is administeredin combination with other therapies suitable for treatment or preventionof neurodegenerative diseases or therapies suitable for relieving thesymptoms of neurodegenerative diseases.

DESCRIPTION OF THE INVENTION 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described can be used in thepractice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “about” refers to a quantity, level, value,dimension, size or amount that varies by as much as 30%, 20% or 10% to areference quantity, level, value, dimension, size or amount.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The term “dephosphorylation” as used herein, refers to the chemicalremoval of a phosphate group (PO₄ ²⁻) from a biochemical entity such asa protein. Under cellular conditions, dephosphorylation is achievedenzymatically by an enzyme such as a phosphatase.

As used herein, the terms “glial cell” and “glial cells” refer tonon-neuronal cells that provide structural and metabolic support forneurons in the central nervous system. Glial cells may also be referredto as neuroglia or glia.

The term “hyperphosphorylation” refers to the circumstance where allavailable phosphorylation sites on a biochemical entity such as aprotein, are phosphorylated. No further phosphorylation of thebiochemical entity can occur. The phrase “inhibiting or reducinghyperphosphorylation” includes preventing all sites on a biochemicalentity from being phosphorylated and decreasing the number ofbiochemical entities that have all of their phosphorylation sitesphosphorylated.

As used herein, the term “in combination with” refers to the treatmentof a subject with at least two agents such that their effects on theneurodegenerative disease occur, at least in part, over the same timeperiod. Administration of at least two agents may occur simultaneouslyin a single composition, or each agent may be simultaneously orsequentially administered in separate compositions.

The terms “Lewy body” and “Lewy bodies” refer to abnormal aggregates ofprotein that develop in nerve cells. The primary protein aggregate in aLewy body is composed of α-synuclein.

The term “neurodegenerative disease” as used herein, refers to aneurological disease characterised by loss or degeneration of neurons.Neurodegenerative diseases include neurodegenerative movement disordersand neurodegenerative conditions relating to memory loss and/ordementia. Neurodegenerative diseases include tauopathies andα-synucleopathies. Examples of neurodegenerative diseases include, butare not limited to, presenile dementia, senile dementia, Alzheimer'sdisease, Parkinsonism linked to chromosome 17 (FTDP-17), progressivesupranuclear palsy (PSP), Pick's disease, primary progressive aphasia,frontotemporal dementia, corticobasal dementia, Parkinson's disease,Parkinson's disease with dementia, dementia with Lewy bodies, Down'ssyndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS)and Hallervorden-Spatz syndrome.

The term “neurofibrillary tangles” as used herein, refers to abnormalstructures located in the brain and composed of dense arrays of pairedhelical filaments (neurofilaments and microtubules). Neurofibrillarytangles include tau proteins, particularly microtubule-associated tauproteins. The number of neurofibrillary tangles present in a brain isbelieved to correlate with the degree of dementia in the subject.Neurofibrillary tangles are a distinguishing characteristic ofAlzheimer's disease.

As used herein, the term “neuron” refers to cells found in the centralnervous system that are specialised to receive, process and transmitinformation. Neurons may also be referred to as nerve cells.

As used herein, the term “nutritional amount” includes an amount ofselenium that provides an average daily intake. In the United States,the average daily intake is 80-120 μg/day.

By “pharmaceutically salt” as used herein in relation to selenate, meansmetal ion salts which are toxicologically safe for human and animaladministration. For example, suitable pharmaceutically acceptable saltsinclude, but are not limited to, salts of pharmaceutically acceptableinorganic acids such as hydrochloric, sulphuric, phosphoric, nitric,carbonic, boric, sulfamic, and hydrobromic acids, or salts ofpharmaceutically acceptable organic acids such as acetic, propionic,butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric,lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic,methanesulphonic, toluenesulphonic, benzenesulphonic, salicyclicsulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic,lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to, those formed withpharmaceutically acceptable cations, such as sodium, potassium, lithium,calcium, magnesium, iron, nickel, zinc, ammonium and alkylammonium.

Basic nitrogen-containing groups may be quarternised with such agents aslower alkyl halide, such as methyl, ethyl, propyl and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl and diethylsulfate; and others.

Suitable metal ion salts of selenate include, but are not limited to,sodium, potassium, lithium, magnesium, calcium, iron, nickel, zinc,ammonium and alkylammonium salts. In some embodiments the salt is notlithium selenate. A preferred salt of selenate is the sodium salt,Na₂SeO₄.

The term “phosphorylation” as used herein refers to the chemicaladdition of a phosphate group (PO₄ ²⁻) to a biochemical entity such as aprotein. Under cellular conditions phosphorylation is achievedenzymatically by an enzyme such as a kinase. The phrase “inhibiting orreducing phosphorylation” includes preventing phosphorylation of one ormore phosphorylation sites on a biochemical entity, including preventingphosphorylation of all phosphorylation sites as in hyperphosphorylation.This phrase also includes decreasing the extent of phosphorylation of abiochemical entity by preventing phosphorylation occurring at one ormore phosphorylation sites or as a result of dephosphorylation occurringat one or more phosphorylated sites on the biochemical entity.

The terms “subject” or “individual” or “patient”, used interchangeablyherein, refer to any subject, particularly a vertebrate subject and moreparticularly a mammalian subject, for whom prophylaxis or treatment isdesired. Suitable vertebrate animals that fall within the scope of theinvention include, but are not limited to, primates, avian, livestockanimals (e.g. pigs, sheep, cows, horses, donkeys), laboratory testanimals (e.g. rabbits, mice, rats, guinea pigs, hamsters), companionanimals (e.g. cats and dogs) and captive wild animals (e.g. foxes, deer,dingoes). A preferred subject is a human in need of treatment orprophylaxis of a neurodegenerative disease, especially Alzheimer'sdisease or dementia. However, it will be understood that theaforementioned terms do not imply that symptoms are present.

The term “supranutritional” as used herein, refers to an amount which isgreater than the amount considered as a nutritional requirement. In theUnited States, the average daily intake of selenium is 80-120 μg/day. Asupranutritional amount of selenium provides selenium to a subject abovethe recommended daily allowance. For example, a supranutritional amountof selenium may be 3 μg/kg to 20 mg/kg per day, 0.015 mg/kg to 20 mg/kg,0.1 mg/kg to 20.0 mg/kg, 0.1 mg/kg to 14 mg/kg, 0.1 mg/kg to 13 mg/kg,0.1 mg/kg to 12 mg/kg, 0.1 mg/kg to 10 mg/kg, 0.1 mg/kg to 9 mg/kg, 0.1mg/kg to 8 mg/kg, 0.1 mg/kg to 7 mg/kg, 0.1 mg/kg to 6 mg/kg, 0.15 mg/kgto 5 mg/kg, 0.15 mg/kg to 4 mg/kg, 0.15 mg/kg to 3 mg/kg, 0.15 mg/kg to2 mg/kg, 0.15 mg/kg to 1 mg/kg per day, especially 0.1 mg/kg to 14mg/kg, 0.07 mg/kg to 6.5 mg/kg or 0.15 mg/kg to 5 mg/kg per day, moreespecially 0.07 mg/kg to 2 mg/kg per day.

As used herein, the term “α-synucleopathy” refers to a neurodegenerativedisorder or disease involving aggregation of α-synuclein or abnormalα-synuclein in nerve cells in the brain. α-Synucleopathies include, butare not limited to, Parkinson's disease, Parkinson's disease withdementia, dementia with Lewy bodies, Pick's disease, Down's syndrome,multiple system atrophy, amylotrophic lateral sclerosis (ALS) andHallervorden-Spatz syndrome.

As used herein, the term “effective amount” in the context of treatingor preventing a neurodegenerative disease or inhibiting or reducingphosphorylation of tau protein or inhibiting the activity of GSK3β ismeant the administration or addition of an amount of selenate or apharmaceutically acceptable salt thereof, either in a single dose or aspart of a series of doses, that is effective in enhancing the activityof PP2A and especially that is effective for the prevention of incurringa symptom, holding in check such symptoms, and/or treating existingsymptoms, associated with the neurodegenerative disease. The effectiveamount will vary depending on the health and physical condition of theindividual to be treated, the taxonomic group of the individual to betreated, the formulation of the composition, the assessment of themedical situations and other relevant factors. It is expected that theamount will fall within a relatively broad range that can be determinedthrough routine trials. In specific embodiments, a effective amount is anutritional or supranutritional amount.

The term “tauopathy” as used herein refers to a neurodegenerativedisorder or disease involving the deposition of abnormal tau proteinisoforms in neurons and glial cells in the brain. Taopathies includediseases and disorders in which tau proteins are abnormallyphosphorylated, including tau protein which is hyperphosphorylated.Tauopathies include, but are not limited to, presenile dementia, seniledementia, Alzheimer's disease, Parkinsonism linked to chromosome 17(FTDP-17), progressive supranuclear palsy (PSP), Pick's disease, primaryprogressive aphasia, frontotemporal dementia and corticobasal dementia.

2. Methods of Treating or Preventing Neurodegenerative Diseases

The present invention is predicated in part on the determination thatselenate or a pharmaceutically acceptable salt thereof, is effective inenhancing the activity of PP2A which in turn may result in a reductionin phosphorylation of tau protein by GSK3β and/or an increase in therate of dephosphorylation of tau protein. The methods of the inventiongenerally comprise exposing PP2A present in neurons or glial cells to aPP2A activity enhancing amount of selenate or a pharmaceuticallyacceptable salt thereof. Suitably, the PP2A activity enhancing amount ofselenate is a nutritional or supranutritional amount of selenate or apharmaceutically acceptable salt thereof. In some embodiments, theamount of selenate or a pharmaceutically acceptable salt thereof,especially selenate or a salt thereof, is from about 0.015 mg/kg toabout 20 mg/kg, usually from about 0.1 mg/kg to 14 mg/kg, 0.07 mg/kg to6.5 mg/kg or 0.15 mg/kg to 5 mg/kg per day, for example, 0.07 mg/kg to 2mg/kg per day.

The present invention can be used effectively to treat or preventneurodegenerative diseases. Neurodegenerative diseases includeneurodegenerative movement disorders and neurodegenerative diseasesassociated with memory loss and include tauopathies andα-synucleopathies. Illustrative examples of neurodegenerative diseasesinclude presenile dementia, senile dementia, Alzheimer's disease,Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclearpalsy (PSP), Pick's disease, primary progressive aphasia, frontotemporaldementia, corticobasal dementia, Parkinson's disease, Parkinson'sdisease with dementia, dementia with Lewy bodies, Down's syndrome,multiple system atrophy, amyotrophic lateral sclerosis (ALS) andHallervorden-Spatz syndrome. In preferred embodiments, the invention issuitable for treating or preventing tauopathies, especially Alzheimer'sdisease and dementia. In other embodiments, the invention is suitablefor treating or preventing an α-synucleopathy, especially Parkinson'sdisease. Suitably, the effective amount of selenate or apharmaceutically acceptable salt thereof is a nutritional orsupranutrional amount of selenate. In some embodiments, the amount ofselenate or a pharmaceutically acceptable salt thereof, is from about0.015 mg/kg to about 20 mg/kg, usually from about 0.1 mg/kg to 14 mg/kgor 0.07 mg/kg to 6.5 mg/kg or 0.15 mg/kg to 5 mg/kg per day, forexample, 0.07 mg/kg to 2 mg/kg per day. In preferred embodiments, theselenate or a pharmaceutically acceptable salt thereof is sodiumselenate (Na₂SeO₄).

In some embodiments, the selenate or a pharmaceutically acceptable saltthereof is administered to a subject in combination with another therapyfor treating or preventing a neurodegenerative disease. Illustrativeexamples of therapies for treating or preventing a neurodegenerativedisease that may be used in combination with selenate or apharmaceutically acceptable salt thereof include, but are not limitedto, cholinesterase inhibitors such as Tacrine (Cognex®), Donepezil,Galantamine and Rivastigmine; N-methyl-D-aspartate (NMDA) receptorantagonists such as Memantine; estrogen therapies such as Premarin,non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin andibuprofen, levodopa (L-Dopa), dopa decarboxylase inhibitors such asCarbidopa and benserazide, or combinations of L-Dopa and a dopadecarboxylase inhibitor, such as Sinemet® and Stalevo®, dopamineagonists such as bromocriptine (Parlodel®), pergolide (Permax®),pramipexole (Mirapex®), ropinirole (Requip®), cabergoline, apomorphine(APOKYN™) and lisuride, mono-amine oxidase B inhibitors such asselegiline (Eldepryl® and Carbex®) and rasagiline (Azilect®),anticholinergics such as benzotropine mesylate (Cogentin®) andtrihexyphenidyl hydrochloride (Artane®) and COMT inhibitors such asEntacapone (Commtan®) and Tolcapone (Tasmar®), or other medications suchas rivastigmine tartrate (Exelon®) and Amantadine (Symmetrel®); ormixtures of two or more of levodopa, dopa decarboxylase inhibitors,dopamine agonists, mono-amine oxidase B inhibitors, anticholinergics orCOMT inhibitors.

Combination therapies could include effective amounts of selenate or apharmaceutically acceptable salt thereof together with an agent used fortreating or preventing a neurodegenerative disease in an amount normallyused in the absence of selenate. For example, tacrine hydrochloride maybe administered as part of a combination with selenate or apharmaceutically acceptable salt thereof to patients with aneurodegenerative disease such as AD in an amount of 40 mg/day to 160mg/day or donepezil may be administered in an amount of 5-10 mg/day.Premarin may be administered at a dosage to achieve 1.25 mg/day ofconjugated equine estrogens (CEEs) in patients with dementia.Alternatively the amount of agent used in the treatment ofneurodegenerative disorders may be decreased upon co-administration withselenate or a pharmaceutically acceptable salt thereof. In someembodiments, the combination may display a synergistic effect.

Certain embodiments of the present invention are directed to methods fortreating or preventing neurodegenerative diseases in a subject, whichmethods generally comprise administering to the subject an effectiveamount of selenate or a pharmaceutically acceptable salt thereof. Topractice these methods, the person managing the subject can determinethe effective dosage form of selenate or a pharmaceutically acceptablesalt thereof for the particular condition and circumstances of thesubject. An effective amount of selenate is one that is effective forthe treatment or prevention of a neurodegenerative disease, includingprevention of incurring a symptom, holding in check a symptom andtreating a symptom. In some embodiments, the effective amount is anutritional amount. In other embodiments, the effective amount is asupranutritional amount. In specific embodiments, the selenate or apharmaceutically acceptable salt thereof is sodium selenate.

Modes of administration, amounts of selenate administered, and selenateformulations, for use in the methods of the present invention, arediscussed below. The neurodegenerative disease to be treated may bedetermined by measuring one or more diagnostic parameters indicative ofthe course of the disease, compared to a suitable control. In the caseof a human subject, a “suitable control” may be the individual beforetreatment, or may be a human (e.g., an age-matched or similar control)treated with a placebo. In accordance with the present invention, thetreatment of neurodegenerative diseases includes and encompasses withoutlimitation: (i) preventing a neurodegenerative disease in a subject whomay be predisposed to the disease but has not yet been diagnosed withthe disease and, accordingly, the treatment constitutes prophylactictreatment for the neurodegenerative disease; (ii) inhibiting aneurodegenerative disease, i.e., arresting the development of theneurodegenerative disease; or (iii) relieving symptoms resulting fromthe neurodegenerative disease.

The methods of the present invention are suitable for treating anindividual who has been diagnosed with a neurodegenerative disease, whois suspected of having a neurodegenerative disease, or who is known tobe susceptible and who is considered likely to develop aneurodegenerative disease.

In some embodiments of the above methods, the neurodegenerative diseaseis a tauopathy, especially Alzheimer's disease or dementia and thetreatment optionally further comprises administration of another agentsuitable for treating a taupathy as described above.

In other embodiments of the above methods, the neurodegenerative diseaseis an α-synucleopathy, especially Parkinson's disease and the treatmentoptionally further comprises administration of another agent suitablefor treating an α-synucleopathy as described above.

In preferred embodiments, the selenate is sodium selenate.

Exemplary subjects for treatment with the methods of the invention arevertebrates, especially mammals. In certain embodiments, the subject isselected from the group consisting of humans, sheep, cattle, horses,bovine, pigs, dogs and cats. A preferred subject is a human.

The selenate or a pharmaceutically acceptable salt thereof may beformulated by following any number of techniques known in the art ofdrug delivery. Selenate or a pharmaceutically acceptable salt thereofmay of course be administered by a number of means keeping in mind thatall formulations are not suitable for every route of administration.Selenate or a pharmaceutically acceptable salt thereof can beadministered in solid or liquid form. The application may be oral,rectal, nasal, topical (including buccal and sublingual), or byinhalation. Selenate or a pharmaceutically acceptable salt thereof maybe administered together with conventional pharmaceutical acceptableadjuvant, carriers and/or diluents.

The solid forms of application comprise tablets, capsules, powders,pills, pastilles, suppositories and granular forms of administration.They may also include carriers or additives, such as flavors, dyes,diluents, softeners, binders, preservatives, lasting agents and/orenclosing materials. Liquid forms of administration include solutions,suspensions and emulsions. These may also be offered together with theabove-mentioned additives.

Solutions and suspensions of selenate or a pharmaceutically acceptablesalt thereof, assuming a suitable viscosity for ease of use, may beinjected. Suspensions too viscous for injection may be implanted usingdevices designed for such purposes, if necessary. Sustained releaseforms are generally administered via parenteral or enteric means.Parenteral administration is another route of administration of theselenate or a pharmaceutically acceptable salt thereof used to practicethe invention. “Parenteral” includes formulations suitable for injectionand for nasal, vaginal, rectal, and buccal administration.

The administration of selenate or a pharmaceutically acceptable saltthereof may involve an oral prolonged dose formulation. Oral doseformulations are preferably administered once daily to three times dailyin the form of a sustained release capsule or tablet, or alternativelyas an aqueous based solution. Selenate or a pharmaceutically acceptablesalt thereof may be administered intravenously either daily,continuously, once a week or three times a week.

The administration of selenate or a pharmaceutically acceptable saltthereof may include daily administration, preferably once daily in theform of a sustained release capsule or tablet, or once daily as anaqueous solution.

Combinations of selenate or a pharmaceutically acceptable salt thereofand at least one agent that is suitable for treating a neurodegenerativedisease and may be administered in solid or liquid form in a singleformulation or composition or in separate formulations or compositions.In some embodiments, the selenate or a pharmaceutically acceptable saltthereof and the agent for treating a neurodegenerative disease areadministered orally as a single tablet or capsule or separate tablets orcapsules. In other embodiments, the selenate or a pharmaceuticallyacceptable salt thereof and the agent for treating a neurodegenerativedisease are administered intravenously in a single composition orseparate compositions.

The present invention also provides pharmaceutical compositions fortreating or preventing a neurodegenerative disease, comprising anutritional or supranutritional amount of selenate or a pharmaceuticallyacceptable salt thereof. In some embodiments, the compositions containfrom about 0.5 mg to about 1.0 g, for example, 5 mg to 450 mg, ofselenate or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. In some embodiments, the selenateor its pharmaceutically acceptable salt is in an amount of about 5.0 mgto about 700 mg or 5 mg to 450 mg. In illustrative examples, theselenate or a pharmaceutically acceptable salt thereof is an amount ofabout 1.6 mg to 450 mg, 5 mg to 450 mg, 7.5 mg to 250 mg, especially 50mg to 200 mg, for example, 50 mg to 100 mg or 100 mg to 150 mg for asingle or divided daily dose. In some embodiments, the pharmaceuticalcompositions are useful for treating or preventing a taupathy,especially Alzheimer's disease or dementia. In other embodiments, thepharmaceutical compositions are useful for treating or preventing anα-synucleopathy, especially Parkinson's disease.

The pharmaceutical compositions comprising selenate or apharmaceutically acceptable salt thereof may further comprise anotheragent for treating or preventing a neurodegenerative disease. Forexample, the composition may contain selenate or a pharmaceuticallyacceptable salt thereof and a cholinesterase inhibitor such as Tacrine,Donepezil, Galantamine or Rivastigmine, an N-methyl-D-aspartate (NMDA)receptor antagonist such as Memantine, an estrogenic agent such asPremarin or a non-steroidal anti-inflammatory drug (NSAID) such asaspirin or ibuprofen, levodopa, a dopa decarboxylase inhibitor,combinations of levodopa and a dopa decarboxylase inhibitor and/or aCOMT inhibitor, a dopamine agonist, a monoamine oxidase B inhibitor, ananticholinergic, a COMT inhibitor or another medication such asrivastigmine tartrate or Amantadine.

The pharmaceutical composition of the present invention may include anyadditional components that are non-immunogenic and biocompatible withselenate, as well as capable of bioabsorption, biodegradation,elimination as an intact molecule. The formulation may be supplied in aready-to-use form or may be supplied as a sterile powder or liquidrequiring vehicle addition prior to administration. If sterility isdesired, the formulation may be made under sterile conditions, theindividual components of the mixture may be sterile, or the formulationmay be sterile filtered prior to use. Such a solution can also containappropriate pharmaceutically acceptable carriers, such as but notlimited to buffers, salts, excipients, preservatives, etc.

In some embodiments, sustained release oral formulations are used foradministering selenate or a pharmaceutically acceptable salt thereof inthe methods of the invention. These formulations generally compriseselenate or a pharmaceutically acceptable salt thereof having decreasedsolubility in order to delay absorption into the bloodstream. Inaddition, these formulations may include other components, agents,carriers, etc., which may also serve to delay absorption of the selenateor a pharmaceutically acceptable salt thereof. Microencapsulation,polymeric entrapment systems, and osmotic pumps, which may or may not bebioerodible, may also be used to allow delayed or controlled diffusionof the selenate or a pharmaceutically acceptable salt thereof from acapsule or matrix.

The selenate or a pharmaceutically acceptable salt thereof can be usedsolus or as part of another agent. Accordingly, the present inventionalso contemplates an agent that comprises selenate or a pharmaceuticallyacceptable salt thereof for the treatment of a neurodegenerativedisease.

In order that the nature of the present invention may be more clearlyunderstood and put into practical effect, particular preferredembodiments thereof will now be described with reference to thefollowing non-limited examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a comparative representation of the levels of phosphorylatedAkt following treatment both in intact cells and in a cell freeenvironment for PC3 cells.

FIG. 1B is a graphical representation comparing the effect of sodiumselenate and staurosporine, a potent but non-specific protein kinaseinhibitor, on the enzymatic activity of recombinant human Alai usingCalbiochem K-LISA™ Akt activity kit.

FIG. 2A is a comparative representation of Akt phosphorylation at Ser⁴⁷³in the presence of sodium selenate or okadaic acid, a polyether toxinfrom red-tide algae which inhibits phospho-protein phosphatases PP1 andPP2A.

FIG. 2B is a comparative representation of Akt phosphorylation at Ser⁴⁷³in the presence of sodium selenate or a number of phospho-proteinphosphatase inhibitors, tautamycin, okadaic acid, endothall A, calyculinA and cyclosporine A.

FIG. 2C provides representations of immunoprecipitation of Akt from PC3cells showing the amount of Akt complexed with PP2A in the absence(control) and presence of selenate (ATE) or fetal calf serum (FCS).

FIG. 2D is a graphical representation of phosphatase PP2A activity inPC3 cells in the presence or absence of sodium selenate.

FIG. 3 is a schematic representation of the post-translationalregulation of PP2A phosphatase activity and substrate specificity(LCMT—leucine carboxymethyltransferase, PME-1—phosphatase methylesterase1, PTPA—Phospholyrosyl phosphatase activator).

FIG. 4A(i) is a graphical representation of relative PP2A phosphataseactivity in the absence (control) of, or in the presence of sodiumselenate or okadaic acid (OKA).

FIG. 4A(ii) is a graphical representation showing the effects of sodiumselenate on the concentration of inorganic phosphate released from aserine phosphopeptide by the phosphatase action of PP2A.

FIG. 4B is a graphical representation showing the effects of sodiumselenate on the phosphatase activity of PP1.

FIG. 5A is a schematic representation of the inactivation of PP2A in anoxidising environment.

FIG. 5B is a representation showing the effects of sodium selenate andhydrogen peroxide on the level of Akt phosphorylation.

FIG. 5C is a graphical representation of the free sulfhydryl groupspresent in the PP2A phosphatase in the presence of N-ethylmaleimide(NEM), hydrogen peroxide, sodium selenate and sodium selenate togetherwith hydrogen peroxide.

FIG. 5D is a graphical representation of fluorescence distributionhistogram for cells treated with sodium selenate or N-acetyl cysteine(NAC) with or without hydrogen peroxide.

FIG. 5E graphically represents the mean percentage of fluorescent cellsupon treatment with hydrogen peroxide, sodium selenate, hydrogenperoxide and sodium selenate, NAC, and hydrogen peroxide and NAC.

FIG. 6A graphically represents the relative phosphatase activity ofimmunoprecipitated PP2A with sodium selenate (ATE 500 μM) or endothall A(ETA 100 μM) or without an additive (control).

FIG. 6B is a comparative representation of levels of phosphorylatedp7OS6K following treatment with LY294003 (LY 50 μM) or sodium selenate(ATE 500 μM), with our without pre-treatment with okadaic acid (OKA 500nM).

FIG. 6C is a comparative representation of the probing of different Bfamily subunits (B and B′) of PP2A with antibodies that recognize Akt.Akt co-precipitated only with the B family subunit of PP2A.

FIG. 7 is a representation showing the effects of sodium selenate onGSK3β activation.

FIG. 8 is a comparative representation showing the selenomethionine didnot affect the level of Akt phosphorylation whereas selenate inhibitedAkt phosphorylation.

FIG. 9 is a graphical representation showing the effects of differentselenium compounds on activation of Akt. Treatments: control (con);sodium selenate (ATE); Selenous acid (Sel acid); sodium selenite (ITE);selenium dioxide (SeO₂); selenium sulfide (SeS₂); methyl selenocysteine(MSC); and selenocysteine (SeC). Relative active Akt signal intensitycorrelated to total Akt protein levels is depicted on the y-axis. Thegraph indicates that only sodium selenate (ATE) inhibits activation ofAkt, reducing levels of phosphorylated Akt below control (con) levels.In contrast, selenous acid (Sel acid), sodium selenite (ITE), seleniumdioxide (SeO₂), selenium sulfide (SeS₂), methyl selenocysteine (MSC),selenocysteine (SeC) all induce activation of Akt above control (con)levels.

FIGS. 10A and 10B graphically represent the effect of sodium selenate(ATE), sodium selenite (ITE) and selenomethionine (SeMet) on PP2Aphosphatase activity in the pNPP hydrolysis assay using pNPP (FIG. 10A)or a serine phosphopeptide (FIG. 10B) as a substrate.

FIG. 11 is a comparative representation of immunoblotting of humanneuroblastoma BE2M17 cell line with anti-human PHF-tau antibodies in thepresence (+) or absence (−) of sodium selenate under various coatingconditions.

FIG. 12 is a comparative representation of immunoblotting of humanneuroblastoma SY5Y cell lines with anti-human PHF-tau antibodies, AT270(FIG. 12A) and HT-7 (FIG. 12B), in the presence (+) or absence (−) ofsodium selenate under various coating conditions.

FIG. 13 is a comparative representation of immunoblotting of total brainlysates from 14 weeks old Balb/c Nu Nu male mice with anti-human PHF-tauantibodies, AT100 (FIG. 13A), AT180 (FIG. 13B), AT270 (FIG. 13C) andHT-7 (FIG. 13D), in the presence (+) or absence (−) of sodium selenate.

FIG. 14 graphically represents the effects of treatment with sodiumselenate on behaviour tested in the Open Field (thigmotaxis) at turn 1(FIG. 14A) and at turn 2 (FIG. 14B).

FIG. 15 graphically represents the effects of treatment with sodiumselenate on learning and memory tested in the Morris Water Maze task asassessed by escape latency (time in seconds, FIG. 15A) and swimming path(length in meters, FIG. 15B).

FIG. 16 graphically represents the effect of sodium selenate on Tau loadin the hippocampus (FIG. 16A) and in the amygdala (FIG. 16B) asdetermined by HT7 Immunohistochemistry in hTAU441 transgenic TMHT mice.

FIG. 17 graphically represents the effect of sodium selenate on Tau loadin the hippocampus (FIG. 17A) and in the amygdala (FIG. 17B) asdetermined by AT180 immunohistochemistry in hTAU441 transgenic TMHTmice.

EXAMPLES Example 1 Sodium Selenate Dephosphorylates Akt by an IndirectMechanism

Sodium selenate consistently induces the dephosphorylation of Akt inintact prostate carcinoma cells. Such dephosphorylation may be theresult of a direct inhibitory effect on the Akt protein itself, oralternatively, can be similarly achieved indirectly by boosting anegative regulator or inhibiting a positive regulator of Aktphosphorylation. To distinguish between a direct and an indirectmechanism, the effect of sodium selenate on Akt phosphorylation andactivity in a cell free environment was determined. PC3 prostatecarcinoma cells were plated in 100 mm dishes, and when 70-80% confluentserum starved overnight. To determine the effect on Akt phosphorylationin intact cells, PC3 cells were treated with sodium selenate 500 μM infresh serum free media for 1 hour. Cells were lysed, and the level ofAkt Ser473 phosphorylation determined by immunoblot analysis with anactivation specific antibody, and comparison made to levels of Aktprotein expressed. To determine the effect on purified Akt, similarlyplated but untreated PC3 cells were lysed in RIPA (minus phosphataseinhibitors), a stringent cell lysis buffer that minimizes maintenance ofprotein-protein complexes. Akt was purified from equal amounts (40-500μg) of whole cell lysate (WCL) by immunoprecipitation with a pan-Aktmonoclonal antibody, and then incubated with 500 μM sodium selenate for1 h in a heat block at 37° C. The immunoprecipitated protein was thenresolved and the level of activated Akt determined by immunoblotanalysis as described above. FIG. 1A compares the levels ofphosphorylated Akt following treatment both in intact cells and a cellfree environment for PC3 cells. Treatment of intact PC3 cells with 500μM sodium selenate for 1 h results in a profound reduction in Aktphosphorylation.

To confirm these findings, the effect of sodium selenate on theenzymatic activity of recombinant human Akt1 was measured using theCalbiochem K-LISA™ Akt activity kit, and compared it to the effect ofstaurosporine, a potent but non specific protein kinase inhibitor. ThisELISA based assay uses a biotinylated peptide substrate (GRPRTSSFAEG)that is phosphorylated on the second serine by Akt. 250 ng ofrecombinant human Akt1 with or without sodium selenate 500 μM orstaurosporine 1 μM was incubated with biotinylated-Akt substrate instreptavidin-coated wells for 30 min at 30° C. Bound phosphorylatedsubstrate was then detected with a phospho-serine detection antibody,followed by the HRP-antibody conjugate, and colour development with TMBsubstrate. Absorbance measured at 450 nm with reference to 590 nm. Theresults of three independent experiments, summarised as mean calculatedabsorbance (A₄₅₀−A₅₉₀−blank)±SEM, are shown in FIG. 1B. Consistent withthe observations described above, sodium selenate had no measurabledirect effect on the kinase activity of recombinant human Akt. Incontrast, the non-specific inhibitor staurosporine potently inhibitedthe kinase activity of Akt.

In summary, these data indicate the sodium selenate has no directinhibitory effect on Akt activity, indicating that the observeddephosphorylation must be via an indirect mechanism.

Example 2 Sodium Selenate Dephosphorylates Akt by Stimulating thePhosphatase Activity of PP2A

A reduction in net Akt activity could be achieved indirectly by eitherpreventing the initial Akt phosphorylation, or alternatively, increasingAkt dephosphorylation [Kohn et. al. 1996]. However, given the biphasicresponse of Akt phosphorylation (an initial transient boost followed bya profound and sustained decrease) induced by sodium selenate, it seemedunlikely that sodium selenate was acting by simply decreasing theability of an upstream kinase to phosphorylate Akt. The effect ofprotein phosphatase inhibition on the ability of sodium selenate toinduce Akt dephosphorylation was therefore determined. Initially okadaicacid, a polyether toxin from red-tide algae (and causative agent ofdiarrhoetic shellfish poisoning) which inhibits the phospho-proteinphosphatases PP1 and PP2A, two phosphatases implicated in the regulationof Akt dephosphorylation was used [Fernandez et al., 2002]. 5×10⁵ PC3prostate carcinoma cells were plated per well of a 6 well plate, allowedattach for 8 h, and then serum starved overnight. Cells were pretreatedwith or without okadaic acid 5 nM or 1000 nM for 30 min in fresh serumfree media, and then sodium selenate added to a final concentration of500 μM for 1 h. Lysed cells were resolved by SDS-PAGE, then probed forAkt phosphorylation at the Ser473 residue by immunoblot analysis with anantibody that specifically recognizes Aid when phosphorylated at thissite, and comparison made with the total levels of Akt protein expressed(FIG. 2A). Treatment of the PTEN deficient PC3 cells with sodiumselenate, as expected, markedly reduces the chronically high levels ofAkt phosphorylation observed in this cell line, and this reduction isunaffected by prior incubation with 5 nM okadaic acid. In contrast,pre-treatment with 1000 nM of okadaic acid not only boosts Aktphosphorylation in non-selenate controls, but completely abolishes theinhibitory effect of sodium selenate.

To further refine the phosphatase involved, a broad panel ofphospho-protein phosphatase (PPP) inhibitors, which differ in theirspecificities for PPP inhibition, were screened for their ability toabrogate the inhibitory effect of sodium selenate on Aktphosphorylation. This panel included tautamycin 500 nM (PP1), okadaicacid 500 nM (low dose PP2A>PP1), endothall A (PP2A) 100 μM, calyculin A2 nM (low dose PP1>PP2A), calyculin A 10 nM (high dose inhibits PP1 andPP2A) and cyclosporine A 400 ng/ml (PP2B). PC3 prostate carcinoma cellswere plated out essentially as described above, and similarly treatedwith sodium selenate 500 μM for 1 h with or without pretreatment with aPPP inhibitor for 30 min prior. Again, lysed cells were resolved bySDS-PAGE, then membranes probed for Akt phosphorylation at the Ser⁴⁷³residue by immunoblot analysis with an antibody that specificallyrecognizes Akt when phosphorylated at this site, and comparison madewith the total levels of Akt protein expressed. As indicated in FIG. 2B,in cells that received no pretreatment with a PPP inhibitor, or in cellsthat were treated with PPP inhibitors that were specific for PP1 orPP2B, exposure to sodium selenate resulted in a marked decrease in thephosphorylation of Akt. In contrast, cells that were treated either withlow dose okadaic acid or endothall A, specific or relatively specificinhibitors of PP2A, completely blocked sodium selenate induced Aktdephosphorylation.

Sodium selenate could increase the PP2A mediated Akt dephosphorylationeither by increasing the rate of complex formation between PP2A and Akt,increasing the intrinsic phosphatase activity of PP2A already bound toPP2A, or both. To help distinguish between these mechanisms it was firstdetermined if treatment with sodium selenate increases the level ofcomplex formation between PP2A and Akt. 1×10⁶ PC3 prostate carcinomacells were plated onto 6 cm dishes, allowed attach for 8 h, and thenserum starved overnight. Cells were then treated either with 500 μMsodium selenate in fresh serum free media, or 10% FCS, for 1.5 h, andthen lysed in ELB buffer. Total Akt was immunoprecipitated from 400 μgof each whole cell lysate using a monoclonal anti-Akt antibody, andcaptured with protein A-sepharose beads. Negative control lysates(blank) had the immunoprecipitating antibody omitted. Following washingto reduce non-specific binding, the beads were boiled for 5 min in 3×SDS protein loading buffer, centrifuged at high speed and thesupernatant resolved by SDS-PAGE. The level of PP2A binding wasdetermined by immunoblot analysis with an antibody that specificallyrecognizes the catalytic subunit of the phosphatase, and comparison madewith the amount of Akt pulled down and the concentration ofprecipitating antibody (IgG). As indicated in FIG. 2C,immunoprecipitation of Akt from untreated PC3 cells increases the amountof PP2A catalytic subunit detectable above that of non-specific bindingto the beads (blank), indicating that even in the basal state with highlevels of Akt phosphorylation, at least some Akt is complexed with PP2A.Treatment with sodium selenate increases the association of PP2Acatalytic subunit with Akt, whereas stimulation with serum decreasescomplex formation to below basal levels. To quantify this increase incomplex formation, representative immunoblots from three independentexperiments were digitalized, subjected to densitometric analysis, andnormalized to the control for each experiment. The mean ratio of PP2Acatalytic subunit to total immunoprecipitated Akt protein±SEM was thendetermined. As indicated in FIG. 2C, an approximately 50% increase inthe binding of PP2A catalytic subunit to Akt protein following treatmentwith sodium selenate was observed.

To determine if the effects of sodium selenate on Akt dephosphorylationcould be fully explained by a simple increase in association between thetwo proteins, the effect of sodium selenate on Akt-associatedphosphatase activity was measured. 2×10⁶ PC3 cells were plated in 10 cmplates, allowed to attach for 8 hours, and then serum starved overnight. Cells were then treated with or without sodium selenate 500 μMfor 1 h in fresh serum free media, then lysed in a low phosphate buffer.500-600 μg of total protein was then immunoprecipitated with anti-Akt(1:100) monoclonal antibody and 30 μl of Protein A slurry, and afterwashing, checked for free phosphate contamination by incubation of 25 μlof final wash buffer with Malachite Green. Immunoprecipitates wereassayed for phosphatase activity by incubation with 500 μM syntheticphospho-threonine peptide for 10 min at 30° C. with agitation. Freephosphate was detected by the addition of malachite green solution, andabsorbance was read at 590 nm for each sample in duplicate. The meanphosphatase activity from three independent experiments±SEM is shown inFIG. 2D. Treatment of PC3 cells with sodium selenate more than doubledthe phosphatase activity associated with immunoprecipitated Akt protein,significantly greater than the simple increase in PP2A bindingpreviously observed.

In summary, these data demonstrate that sodium selenate induces Aktdephosphorylation indirectly through a phospho-protein phosphatase,specifically PP2A. Although sodium selenate increases the amount of PP2Abinding to Akt, the relative increase in associated phosphatase activityis nearly twice that, indicating that sodium selenate may primarilyaffect enzyme activity.

Example 3 Sodium Selenate Directly Boosts the Phosphatase Activity ofPP2A Core Dimer

The core structure of PP2A consists of 36 kDa catalytic subunit (PP2Ac)and a 65 kDa regulatory subunit (PR65 or A subunit). Binding with athird regulatory B subunit regulates substrate specificity [Wera andHemmings, 1995]. PP2A phosphatase activity can be regulated bypost-translational modification, represented schematically in FIG. 3.PP2Ac has been shown to be phosphorylated in vitro by both receptor andnon-receptor tyrosine kinases such as EGFR, the insulin receptor,p60v-src and p56lck [Chen et al., 1992]. This phosphorylation occursspecifically at Tyr307, and is associated with a greater than 90% lossin phosphatase activity [Chen et al., 1992]. This phosphorylation isalso identified in vivo, and is increased in fibroblasts stimulated withserum or EGF, or transformed with p60v-src, whereas it is decreased byserum starvation [Chen et al., 1994]. Phosphorylation of the adjacentThr304 has also been associated with a significant loss of phosphataseactivity [Guo and Damuni, 1993]. In contrast to Tyr307, it appears thatThr304 is phosphorylated by an autophosphorylation-activated proteinkinase, thereby amplifying the initial inhibitory signal. In both cases,PP2A acts as its own phosphatase, as pharmacological inhibition withokadaic acid or microcystin-LR increases phosphorylation [Chen et al.,1994, Guo and Damuni, 1993]. Thus, following the removal of theinhibitory stimulus, PP2A hydrolyses both phosphate groups to rapidlyregenerate active phosphatase. PP2Ac is also subject to regulation byreversible methylation of a carboxy terminal lysine residue, L309. Themethylation reaction is catalysed by a leucine carboxylmethyltransferase [Xie and Clarke, 1994], and appears necessary for thecorrect association of subunits to form an active trimer [Wu et al.,2000, Tulstylch et al., 2000, Bryant et al., 1999]. PP2Ac isdemethylated by phosphatase methylesterase 1 (PME-. 1) [Lee et al.,1996]. Interestingly, PME-1 has also been reported to bind to andpotentially stabilize PP2a dimers and trimers in an inactiveconformation, a situation reversed by phosphotyrosyl phosphataseactivator (PTPA), a protein originally identified stimulating PP2Atyrosyl phosphatase activity [Cayla et al., 1994, Langin et al., 2004,Van Hoof et al., 2005].

To determine if the boost in PP2A phosphatase activity observed withsodium selenate was independent of an effect on upstream componentsinvolved in post-translational regulation, the enzymatic activity ofhuman PP2A A-C heterodimer purified from red blood cells incubated inthe presence or absence of sodium selenate was determined. In initialassays a chemical substrate of phosphatase enzymatic action,para-nitrophenylphosphate (pNPP) was used, which following hydrolysis ofthe phosphate moiety generates para-nitrophenol, an intensely yellowchromogen which is soluble under alkaline conditions. 0.05 U of purifiedhuman PP2A dimer was incubated in the presence of 5 mM sodium selenateor 500 nM okadaic acid for a total of 30 min at 37° C., and measured theamount of para-nitrophenol generated, as a readout of phosphataseactivity, compared to untreated control samples. Absorbance of eachsample was measured in duplicate at 405 nm with 590 nm as a reference.PP2A activity was calculated using the following equation:

activity=(sample vol in litres)×A ₄₀₅/1.78×10₄M⁻¹cm⁻¹(extinctioncoefficient)×0.25 cm×15 minutes×0.05 U enzyme

Data is presented as mean relative phosphatase activity±SEM from atleast three independent experiments. As indicated in FIG. 4A(i), evenwith such a low concentration of purified enzyme, phosphatase activitywas readily apparent, and this was completely abolished by incubationwith okadaic acid. In contrast, incubation of PP2A with sodium selenatenearly tripled the observed phosphatase activity.

It was next determined if this boost in phosphatase activity wassubstrate specific by measuring the effect of sodium selenate on theliberation by PP2A A-C dimer of inorganic phosphate from a synthetic 6amino acid peptide, phosphorylated on an internal threonine residue.0.01-0.05 U of PP2A was incubated with 500 μM of phosphopeptide for 15min at 37° C., with or without 50 μM sodium selenate. The amount ofinorganic phosphate released by enzymatic activity was determined by theaddition of malachite green, and absorbance read at 590 nm. Malachitegreen forms a stable green complex in the presence of molybdate andorthophosphate, allowing the concentration of inorganic phosphatepresent to be measured. Data is presented as mean absorbance at 590 nmfrom at least three independent experiments, ±SEM. As indicated in FIG.4A(ii), sodium selenate again more than doubled the concentration ofinorganic phosphate released from the serine phosphopeptide by thephosphatase action of PP2A.

The catalytic subunit of PP1 shares approximately 50% sequence homologywith the catalytic subunit of PP2A, the highest of any of the relatedphosphatases [Barton et al., 1994]. To determine if the boost inphosphatase activity stimulated by sodium selenate was specific to PP2A,its effect on the activity of PP1 was determined. 0.05 U of rabbit PP1purified from skeletal muscle was incubated with 500 μMphospho-threonine synthetic peptide for 15 min at 37° C., with orwithout 50 μM sodium selenate, and the concentration of free phosphatedetermined using malachite green as described above. Data is presentedas mean absorbance at 590 nm from three independent experiments, +SEM.As indicated in FIG. 4B, sodium selenate did not affect the phosphataseactivity of PP1.

Example 4 Sodium Selenate does not Affect the Redox Regulation of PP2A

An increasing body of work suggests that reversible oxidation is acommon mechanism by which protein phosphatases are negatively regulated[Wang et al., 1996, Barrett et al., 1999, Sohn and Rudolph 2003]. Aconserved cysteine residue in the catalytic domain is critical to theirenzymatic activity, but in an oxidating microenvironment this may bemodified by the formation of either intramolecular disulphide orsulphenyl-amide bonds (FIG. 5A), with the loss of phosphatase activity[Salmeen et al., 2003, Kwon et al., 2004]. Given that selenium compoundscan affect cellular redox state, the possibility that sodium selenatestimulated PP2A by relieving the inhibitory effects of oxidation wasinvestigated.

Initially it was determined if the dephosphorylation of Akt induced bysodium selenate was sensitive to the cellular redox state. 5×10⁵ PC3prostate carcinoma cells were plated per well in a 6-well plate, allowedto attach for 8 h, and then serum starved overnight. Cells were treatedwith or without 500 μM sodium selenate in fresh media, and then exposedto hydrogen peroxide at 0.25 mM or 1 mM for 10 mM. Equal amounts ofwhole cell lysates were resolved, and then subjected to immunoblotanalysis to determine the level of phosphorylated Akt Ser⁴⁷³. Comparisonwas then made with the total level of Akt protein expressed. Asindicated in FIG. 5B, treatment of cells with sodium selenate markedlyreduced the level of Akt phosphorylation, and this was unaffected by theacute addition of 0.25 mM of hydrogen peroxide. In contrast, acuteexposure to a higher dose of 1 mM hydrogen peroxide completely abolishedthe dephosphorylating effect of sodium selenate, indicating that thisblock is redox sensitive.

Inactivating reversible oxidation of protein phosphatases involves themodification of a critical cysteine residue within the catalytic domain,ultimately leading to the formation of either intramolecular disulphideor sulphenyl-amide bonds. To determine if sodium selenate had anymodifying effect on cysteine residues in PP2A, the number of freesulfhydryl groups in purified human PP2A A-C dimer was quantifiedfollowing various treatments using Ellman's assay [Ellman, 1958].Ellman's reagent (5,5′-Dithio-bis(2-nitrobenzoic acid), DTNB) rapidlyforms a disulphide bond with free sulfhydryl groups with the release ofchromogenic thiolate ions. 0.01 U of PP2A was incubated withN-ethylmaleimide (NEM) 10 mM or hydrogen peroxide 10 mM or sodiumselenate 10 mM, or sodium selenate 10 mM and hydrogen peroxide 10 mM for15 min at 37° C. The quantity of free sulfhydryl groups present withinthe phosphatase was then determined by the addition of Ellman's reagentand subsequent measurement of absorbance at 412 nm (FIG. 5C). Incubationwith both NEM, a sulfhydryl alkylating agent, and hydrogen peroxidesignificantly reduced the number of free sulfhydryl groups present. Incontrast, incubation with sodium selenate has no effect, and inparticular, did not protect PP2A from hydrogen peroxide mediatedmodification of sulfhydryl groups. Indeed, the modification ofsulfhydryl groups by hydrogen peroxide was significantly more efficientin the presence of sodium selenate.

Next it was determined if sodium selenate affected the redox potentialof intact cells using 2′,7′-dichlorodihydrofluorescein diacetate(DCFDA). DCFDA is non-fluorescent and freely cell permeable, but in thepresence of reactive oxygen species (ROS) is rapidly converted to thecell impermeable but highly fluorescent 2′,7′-dichlorofluorescein (DCF)[Bass et al., 1983]. 1×10⁶ PC3 prostate carcinoma cells were plated in60 mm dishes, allowed to attach for 8 h, and serum starved overnight.Cells were incubated with DCFDA 5 μM for 15 min prior to treatment.

Cells were treated with either 500 μM of sodium selenate or 1 mM ofN-acetylcysteine (NAC) for 1 h, and then exposed to 500 μM of hydrogenperoxide for 10 min. The proportion of fluorescent cells was thendetermined by flow cytometry. FIG. 5D shows a representativefluorescence distribution histogram for each treatment group, and themean percentage of fluorescent cells from three independentexperiments+SEM is summarized in FIG. 5E. Exposure of PC3 cells tohydrogen peroxide lead predictably to a marked increase in thegeneration of intracellular oxygen free radicals, indicated by a shiftto the right in the distribution histogram. Pretreatment of the cellswith sodium selenate had no effect on the basal proportion offluorescent cells, and did not protect the cells from the generation ofROS following exposure to hydrogen peroxide. In contrast, pretreatmentwith the hydrogen donor NAC lead to a small reduction in the basalproportion of fluorescent cells, and in particular, significantlyattenuated ROS production by hydrogen peroxide.

In summary, these data indicate that although the dephosphorylation ofAkt induced by sodium selenate is sensitive to the cellular redox state,sodium selenate does not boost the phosphatase activity of PP2A byrelieving intrinsic but reversible inhibitory oxidation.

Example 5 Sodium Selenate Stimulated PP2A Phosphatase ActivityDemonstrates Substrate Specificity

PP2A is a ubiquitous and highly expressed protein that is estimated tomake up between 0.1-1% of total cellular protein [Gallego M and Virshup2005; Cohen, 1997] and has been implicated in the regulation of an everincreasing number of protein substrates [Zhu et al., 2004; Woetmann etal., 2003; Silverstein et al., 2002]. The mechanism which controls PP2Asubstrate specificity is incompletely understood, but differentialbinding of specific regulatory B subunits appears to be important [VanKanegan et al., 2005]. It was determined if the increased PP2Aphosphatase activity stimulated by sodium selenate was indiscriminate,or specific to a particular heterotrimer.

It has been demonstrated above that sodium selenate increases thephosphatase activity associated with Akt immunoprecipitated from PC3prostate carcinoma cells, and that sodium selenate directly stimulatesthe phosphatase activity of purified PP2A A-C dimer. To determine ifthis boost in phosphatase activity is generalized, PP2A wasimmunoprecipitated from PC3 cells treated with either sodium selenate500 μM or endothall A 100 μM for 1 hour using a monoclonal antibody tothe PP2A catalytic subunit. Free phosphate was removed by passing celllysates through desalting columns, and measured phosphatase activityusing a phosphor-serine peptide and malachite green. The relativephosphatase activity of immunoprecipitated PP2A under the differentconditions is shown in FIG. 6A as the mean absorbance of threeindependent experiments+SEM. The treatment of PC3 cells with sodiumselenate had no effect on the phosphatase activity of the general poolof intracellular PP2A. In contrast, treatment with the PP2A specificphosphatase inhibitor endothall A significantly reduced phosphataseactivity.

The effect of sodium selenate on another known substrate of PP2A, p70S6Kwas examined [Peterson et al., 1999]. 5×10⁵ PC3 prostate carcinoma cellswere plated per well of a 6-well plate, allowed to attach for 8 hours,then serum starved overnight. Cells were then treated with eitherLY294003 50 μM or sodium selenate 500 μM for 1 hour, with or withoutpre-treatment with okadaic acid 500 nM for 30 minutes prior. Equalamounts of whole cell lysates (75 μg) were resolved by SDS-PAGE, and thelevel of p70S6K phosphorylation determined by immunoblot analysis withan antibody that specifically recognizes the p70S6K protein whenphosphorylated on Thr389. Comparison is made with the protein loadingcontrol β tubulin. As indicated in FIG. 6B, even under basal conditions,p70S6K phosphorylation is readily apparent, and this is abolished bytreatment with the PI3K inhibitor LY294002. Consistent with its knownrole as a negative regulator of p70S6K phosphorylation, inhibition ofPP2A with okadaic acid boosts the level of phosphorylation observed.However, in contrast to the dephosphorylation of Akt induced by sodiumselenate, the phosphorylation of p70S6K is unaffected in similarconditions.

The primary determinant of substrate specificity appears to reside inthe regulatory B subunit, which along with the A and catalytic subunits,comprises the trimeric complex observed in vivo.

An attempt was made to determine which family of B subunits formed acomplex with Akt in prostate carcinoma cells. 2×10⁶ PC3 prostatecarcinoma cells were plated into 100 mm dishes, allowed to attach for 8hours, and serum starved overnight. Cells were then lysed with ELB, amild detergent buffer, and total Akt immunoprecipitated from 500 μg oflysate with a pan-Akt monoclonal antibody (1:100) and 30 μL proteinA-sepharose. Negative controls (blank) had the immunoprecipitatingantibody omitted. Following repeated washing the beads were boiled in 3×SDS protein loading buffer for 5 minutes, centrifuged at high speed, andthe resulting supernatant resolved by gel electrophoresis. 100 μg ofwhole cell lysate was run out on the same gel for comparison, and theresulting membranes probed with antibodies that specifically recognizemembers of either B or B′ families of subunits. Successful pulldown ofAkt was confirmed by probing the same blots with an antibody thatrecognizes pan-Akt. As shown in FIG. 6C only a B family regulatorysubunit co-precipitated with Akt in this system, suggesting that atrimer of PP2Ac, A and member of the R2 B subunit family mediates Aktdephosphorylation in these cells.

Example 6

The metabolic effects of insulin on glycogen synthesis are mediated byglycogen synthase kinase-3 (GSK-3), a direct downstream substrate ofAkt. GSK-3 is a ubiquitously expressed serine/threonine kinase thatphosphorylates and inactivates glycogen synthase. In response toactivation of the insulin receptor, Akt phosphorylates and inactivatesthe repressor GSK-3, thereby stimulating glycogen synthesis [Cross etal., 1995]. In addition, GSK-3 has been implicated in the control ofprotein translation, cell cycle progression and Wnt signalling [Diehl etal., 1998, Welsh et al., 1996, He et al., 1995]. To determine the effectof sodium selenate treatment on GSK-3β activation, PC3 prostatecarcinoma cells were plated, serum starved overnight, then treated withsodium selenate 500 μM in fresh media for various times as indicated(FIG. 7). Resolved whole cell lysates were subjected to immunoblotanalysis with an antibody that specifically recognizes GSK-3β only whenphosphorylated at Ser9, a site critical for its kinase activity, andcomparison made with the cytoskeletal protein β-tubulin as a loadingcontrol. PC3 cells have high basal levels of GSK-3β activation,indicated by the high degree of phosphorylation observed in the controllane (0 h). Treatment with sodium selenate led to a marked reduction inphosphorylation, which was maximal at 1 h and 3 h, and returned towardsbasal levels at the 6 h timepoint. To quantify the degree of inhibitionthat sodium selenate induced, digitalized immunoblots were preparedcomparing the effects of sodium selenate 500 μM for 3 h to LY294002 50μM for 1 h and determined the degree of GSK-3β phosphorylation as aproportion of expressed protein by densitometry. On average, sodiumselenate reduced GSK-3β by over 20%, whereas LY294002 resulted in analmost 60% reduction in phosphorylation levels.

Example 7 Sodium Selenate, but not Selenomethionine, Potently InhibitsAkt/Protein Kinase B Activation

To determine whether sodium selenate can interfere with the activity ofthe PI3K pathway, serum free PC3 cells were treated with 500 μM sodiumselenate for various times. The phosphorylation status of Akt in wholecell lysates was determined using activation specific antibodies.Because of the loss of PTEN, even in the absence of additional serum,there is robust activation of Akt at both the Ser473 and Thre308 sites.Addition of sodium selenate induced a transient boost in phosphorylationof Akt at both sites within 10 minutes of exposure. This boost was thenfollowed by a marked and prolonged deactivation, whilst total cellularAkt levels (pan Akt) remained essentially unchanged.

The effect of sodium selenate on Akt activation in PC3 cells wascompared to that of selenomethionine, by treating with 500 μM of eachcompound for various timepoints and assessing Akt phosphorylation atSer473. As indicated in FIG. 8, selenomethionine did not affect thelevel of phosphorylation of Akt at all time periods measured, whereassodium selenate profoundly inhibited Akt phosphorylation.

Example 8 The Ability to Induce Akt Dephosphorylation is Unique toSodium Selenate

Given that sodium selenate consistently induced profounddephosphorylation of the key kinase Akt, whilst selenomethionine had noeffect, it was determined if this ability was unique to sodium selenateor was shared by other chemical forms of selenium. Other inorganic(sodium selenite, selenium dioxide, selenium sulphide and seleniousacid) and organic (methylselenocysteine, selenocystine) selenium specieswere tested for their ability to affect Akt activation. All forms weredissolved in water with the exception of selenium sulphide which wassolubilised in DMSO, and added to serum starved PC3 cells at aconcentration of 500 μM for 1 hour. Whole cell lysates were thenresolved, and the activation status of Akt determined by immunoblottingwith an antibody that specifically recognizes Akt when phosphorylated atthe Ser473 site, as well as the total level of Akt expression (pan Akt).To quantify the degree of Akt activation, immunoblots were digitalised,and the ratio of phosphorylated Akt to total Akt determined bydensitometry. The mean ratio of phosphorylated Akt to total Akt proteinlevels±SEM from three independent experiments summarized in FIG. 9.Treatment of PC3 cells with sodium selenate at 500 μM for 1 hourresulted in a greater than 80% reduction in Akt phosphorylation atSer473 compared to untreated cells (p<0.05, Students t-test). Incontrast, treatment of PC3 cells with other inorganic and organic formsof selenium administered under identical conditions had no significanteffect on the degree of Akt activation. In summary, these data indicatethat the ability to induce Akt dephosphorylation is unique to sodiumselenate.

Example 9 The Ability to Enhance PP2A Activity is Unique to SodiumSelenate

In Example 7, data demonstrating of all of the chemical forms ofselenium tested, sodium selenate alone significantly induceddephosphorylation of Akt was presented. To determine if a differentialeffect on PP2A activity could explain this specificity, the effect ofsodium selenate (5 mM or 50 μM), sodium selenite (5 mM or 50 μM) andselenomethionine (5 mM or 50 μM) on PP2A phosphatase activity wascompared in the pNPP hydrolysis assay using pNPP and a serinephosphopeptide as a substrate respectively (FIGS. 10A and 10B). Incontrast to the clear boost in phosphatase activity observed with sodiumselenate, neither sodium selenite nor selenomethionine significantlyaffected PP2A phosphatase activity.

Materials and Methods for Examples 1 to 9 Reagents Cell Lines

The details of the mammalian cell line used in the experimentalprocedures is given in Table 1.

TABLE 1 Cell Line Origin Source Reference Pc3 Derived from bonemetastasis of a ATCC (Kaighn M E grade IV prostatic adenocarcinoma etal, 1979) from a 62-year-old male Caucasian

All cell cultures were maintained in a Forma Scientific Incubator with5% or 10% carbon dioxide at 37° C. in RPMI 1641 with L-Glutamine (GibcoInvitrogen #11875-119). Penicillin (100 U/ml), streptomycin (100 μg/ml)and amphotericin B (25 ng/ml) (Gibco Invitrogen #15240-062) were addedto media as standard. As a routine, cells were maintained in thepresence of 5% or 10% fetal bovine serum (Gibco Invitrogen #10099-141)unless otherwise stated. Subconfluent cells were passaged with 0.5%trypsin-EDTA (Gibco Invitrogen #15400-054).

Commercially Available Antibodies

A number of commercially available primary and horseradish peroxidase(HRP) conjugated or fluorescently labelled or biotinylated secondaryantibodies were used in the experimental work described, the details ofwhich are summarized below:

Anti-Akt antibody rabbit polyclonal, Cat No. 9272, Cell SignallingTechnology (CST);

Anti-Akt (5G3) mouse monoclonal, Cat No. 2966, CST;

Anti-phospho Akt (ser473) rabbit polyclonal, Cat No. 9271, CST;

Anti-phospho GSK3β (ser9) rabbit polyclonal, Cat No. 9336, CST;

different anti-PP2A antibodies, Cat Nos. 05-421, 06-2221, 07-334 and05-592, Upstate.

Kinases and Kinase Inhibitors, Phosphatase Inhibitors and PurifiedPhosphatases

TABLE 2 kinase and phosphatase inhibitors Enzyme Stock Working Nameinhibited Source Cat No. Diluent Conc. Conc. Kinase LY294002 PI2KPromega V1201 DMSO 50 mM 10-50 μM inhibitor Phosphatase tautomycin PP1Biomol 109946- ethanol 500 μM 500 μM inhibitor 35-2 Phosphataseendothall PP2A Calbiochem 324760 H₂O 100 mM 100 μM inhibitor Phosphatasecalyculin A PP2A = Calbiochem 208821 DMSO 80 μM 10 nM inhibitor PP1Phosphatase okadaic PP2A>> Calbiochem 495604 ethanol 50 mM 500 nMinhibitor Acid PP1 Phosphatase cyclosporin A PP2B Calbiochem 239835 DMSO400 μg/mL 400 ng/mL inhibitor

TABLE 3 purified phosphatases and recombinant kinases Working NameSpecies Source Cat. No. Conc. Purified PP2A Human Upstate 14-111 (lot0.01-0.05 U phosphatase #28002) Purified PP1 Rabbit Upstate 14-110 (lot0.05 U phosphatase #26200) Recombinant Akt1 Human Upstate 14-276 (lotkinase #25089BU)

Buffers, Solutions and Media

All solutions were stored at room temperature (RT) unless otherwisestated.

EDTA 0.5M 186.1 g Na₂EDTA•2H₂0 dissolved in 1 litre dH₂O, pH adjusted to8.0 with NaOH Egg Lysis 250 mM NaCl, 50 mM Hepes pH 7.0, 5 mM EDTA, 1 mMBuffer DTT, 10% Triton-X100 (ELB) NaCl 5M 292.2 g NaCl dissolved in 1litre dH₂O, autoclaved PBS NaCl 8.0 g, KCl 0.2 g, Na₂HPO₄ 1.44 g, KH₂PO₄0.24 g, made up to 1 litre, pH to 7.4 RIPA lysis 10% Glycerol, 20 mMTris-HCl pH 7.5, 2 mM EDTA, 1% buffer Triton-X100, 1% NP40, 137 mM NaCl,0.1% SDS. Made up fresh on ice. SDS 20% 200 g SDS dissolved in 1 litredH₂O SDS-PAGE 0.375 M Tris pH 8.8, 0.1% SDS, APS, TEMED and Running Gel6-14.5% acrylamide SDS-PAGE 15% glycerol, 0.6M 2-mercaptoethanol, 2%SDS, Sample 62.5 mM Tris-HCl pH 6.8 and 0.1% w/v bromophenol Buffer 4xblue SDS-PAGE 0.125M Tris pH 6.8, 0.1% SDS, 4.8% acrylamide Stacking Gel(Biorad), ammonium persulphate (APS) and TEMED SDS-PAGE 9 g Tris base(25 mM) pH 8.3, 43.2 g Glycine Running (0.19M) and 3 g SDS (0.1%) madeup to 500 ml Buffer in dH₂O TBS 10X 80 g NaCl, 2 g KCl, 30 g Tris pH 7.4made up to 1 litre in dH₂O TBS-T 0.1% 500 ml of TBS 10x, 5 ml Tween-20made up to 1 litre in dH₂O Tris-HCl 121.1 g Tris dissolved in 1 litredH₂O, pH adjusted to 6.0-8.8 with concentrated HCl, autoclaved Western14.4 g glycine, 3.03 g Tris, 800 ml dH₂O and 200 ml Transfer methanolBuffer Western 3.03 g Tris base, 14.4 g Glycine in 800 ml dH₂O Transferwith 200 ml methanol Buffer Western 1 ml of 0.5M Tris pH 6.8, 800 μlglycerol, 800 μl Sample 20% SDS, 400 μl β-mercaptoethanol, 400 μl 1%Buffer 4X bromophenol blue

Selenium Compounds

Sodium selenate was purchased from Sigma. All solutions were madefreshly in dH₂O and used at the stated concentrations.

Protein Expression SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Following specified treatments, cells in culture were washed once in PBSand then lysed for 15 min at 4° C. in either Egg Lysis Buffer (ELB) orRIPA lysis buffer containing a protease inhibitor cocktail (CalbiochemProtease Inhibitor Cocktail Set 1 #539131) and phosphatase inhibitors(10 mM Sodium Orthovanadate and 10 mM Sodium Fluoride) with the aid of acell scraper. Samples were clarified by centrifugation at 14,000 rpm for15 min at 4° C. and the supernatant used for analysis. Sample proteinconcentration was determined by Bicinchoninic Acid Solution Assay (BCA).1 μl of lysate was diluted 1:25 with dH₂O in a 96-well plate. 200 μl ofan 80:1 solution of bicinchoninic acid solution and 4% (w/v) coppersulphate was added to each sample and incubated at 37° C. for 30 min.Absorbance was measured at 590 nm (Perkin Elmer MBA2000) against aseries of protein standards.

Protein samples were analysed by gel electrophoresis using denaturingSDS-polyacrylamide gels, consisting of a stacking gel and a resolvinggel. 50-100 μg of protein was loaded on SDS-PAGE gels of varyingconcentrations. SDS sample buffer was added in equal volume to thesamples and boiled at 100° C. for 5 min prior to loading. Gelelectrophoresis was performed in running buffer at approximately 100 V.The molecular weight of proteins of interest was assessed by proteinsize markers (Biorad Kaleidoscope) loaded with each gel.

Western Blot Analysis

Following resolution proteins were transferred to PVDF membrane(Immobilon P, Millipore). The membrane was prepared by immersion in 100%methanol for 10 s, rinsed in distilled water and equilibrated in westerntransfer buffer. Transfer occurred at 100 V at RT for 1.5 h or overnightat 4° C. The membrane was washed for 5 min in TBS-T and allowed to dryfor 1 h. The membrane was then blocked with 3% skim milk in TBS with0.1% Tween for 1 h at RT. The membrane was washed three times for 5 minwith TBS-T and then incubated with the primary antibody diluted in 3%skim milk/0.1% TBS-T or 3% BSA/0.1% TBS-T for 1-2 h at RT or overnightat 4° C. with gentle agitation. Following three 5 min washes in 0.1%TBS-T the membrane was incubated with horseradish peroxidase conjugatedsecondary antibodies in blocking buffer for 1 h. The membrane was againwashed three times for 5 min before antibody binding was detected byenhanced chemiluminescence with Super Signal® West Dura ExtendedDuration Substrate (Pierce #34075) or ECL Western Blotting DetectionAgents (Amersham Biosciences #RPN2106). The luminescence was recorded byautoradiography using CL-exposure film (Kodak). After initial analysisimmunoblots were stripped with Membrane Stripping Buffer (62.5 mM TRISpH7, 2% SDS, 7% β-mercaptoethanol) for 15 min at 60° C., washed threetimes in TBS-T and blocked again before reprobing.

Densitometric Analysis

Relevant immunoblots were converted to .tif files in Corel Photo-PaintVersion 8 (Corel Corporation) using a Vista digital scanner (Umax).Densitometric analysis was performed in Image-Pro-Plus V4.5.1.22 forWindows (Media Cybernetics). Using a linear calibration in which whitewas assigned the value 0 and black the value 255, a region of interestcorresponding to the relevant band was selected and signal intensitydetermined.

Immunoprecipitation

100-600 μg of whole cell lysate was incubated with various antibodies ata 1:100 dilution in microfuge tubes on a rotating wheel for 1-2 h at RTor 16 h at 4° C. as indicated. Following brief centrifugation, 30-40 μlof pre-washed 50% Protein-A Sepharose Fast Flow beads (Sigma #9424) wasadded to each tube and rotated at 4° C. for 1 h at RT or 4° C. Followingbrief centrifugation, the supernatant was removed and the pellet washedwith 500 μl of cell lysis buffer. Following three such washes, beadswere used for enzymatic assays or the protein eluted by boiling thebeads in 3× SDS protein loading buffer for 5 min and resolved onSDS-PAGE gels.

Phosphatase Assays

Purified human PP2Ac and rabbit PP1 were diluted to 0.01 U/μl with thedilution buffer provided and stored in aliquots at −20° C.

Phosphopeptide Assays with Purified Phosphatases

1 mg of synthetic phosphopeptides K-R-pT-I-R-R (Upstate #12-219) andR-R-A-pS-V-A (Upstate #12-220) were dissolved in 1.10-1.285 ml of dH₂Oto prepare a 1 mM solution, then aliquoted and stored at −20° C. untiluse. 0.01-0.05 U of purified PP2A A-C dimer purified from human redblood cells, or 0.05 U of rabbit PP1 purified from skeletal muscle wasmixed with 500 μM of phosphopeptide and incubated in the presence ofvarious treatments as indicated at 37° C. on a heating block for 15 min.Each reaction was made up to a total volume of 25 μl with pNPP buffer(50 mM Tris-HCl, pH 7.0, 100 μM CaCl₂). The enzyme reaction wasterminated by adding 100 μl of malachite green solution (0.034%malachite green in 10 mM ammonium molybdate, 1 N HCl, 3.4% ethanol,0.01% Tween-20). Malachite green forms a stable green complex in thepresence of molybdate and orthophosphate, allowing the amount ofinorganic phosphate present to be measured. Absorbance was read induplicate for each sample at 590 nm.

PP2A Immunoprecipitation and Phosphatase Assay

1×10⁶ PC3 cells were plated in 60 mm plates and allowed attach for 8 h,then serum starved over night. Following various treatments asindicated, the media was aspirated and cells washed once with TBS. Cellswere lysed in 0.3 ml of lysis buffer (20 mM Tris-HCl, pH 7.0, 1%Igepal-CA, 2 mM EDTA, 2 mM EGTA, 1× Complete Protease InhibitorCocktail) and phosphates removed by passing the lysate through a 2 mlZeba Desalt Spin Column (Pierce #89890). Protein concentration of thedesalted lysate was determined by the BCA assay as previously described.100-150 μg of total protein was immunoprecipitated with 4 μg ofanti-PP2A monoclonal antibody and 30 μl of Protein A-sepharose slurry at4° C. for 2 h. Samples were then pelleted at 14,000 rpm for 1 min,washed three times in excess TBS and once with pNPP assay buffer. Afterthe final spin all of the supernatant was carefully removed and 500 μMof phosphor-threonine peptide in a final volume of 80 μl added to thebeads. Samples were incubated at 30° C. for 10 min with agitation, andthe reaction terminated by the addition of malachite green solution.Absorbance was read at 590 nm for each sample in duplicate.

pNPP Hydrolysis Assay

Para-nitrophenylphosphate (pNPP) is a chemical substrate of phosphataseenzymes, which following hydrolysis of the phosphate moiety generatespara-nitrophenol, an intensely yellow chromogen, soluble under alkalineconditions. In assays measuring relative phosphatase activity, 0.05 U ofpurified PP2A was mixed with 5 μl of 40 mM NiCl₂ and 5 μl of BSAsolution (5 mg/ml) in a microcentrifuge tube. Various treatments wereadded as indicated, and the volume adjusted to 80 μl with pNPP assaybuffer. Samples were pre-incubated at 37° C. for 15 min. pNPP substratewas freshly prepared before each assay by dissolving 1.5 mg/ml pNPP in50 mM Tris-HCl, pH 7.0. To start the phosphatase reaction, 120 μl ofpNPP substrate was added, and samples incubated for a further 15 min at37° C. Absorbance of each sample was measured in duplicate at 405 nmwith 590 nm as a reference. PP2A activity was calculated using thefollowing equation: activity=(Sample volume inlitres)×A₄₀₅/1.78×10⁴M⁻¹cm⁻¹ (Extinction coefficient)×0.25 cm×15min×0.05 U enzyme.

Measurement of Total Free Sulfhydryl Groups

The change in free sulfhydryl groups in purified PP2A following varioustreatments was determined using Ellman's Reagent. To 0.01 U of PP2A wasadded to 37.5 μl of dilution buffer (30 mM Tris-HCl, 3 mM EDTA, pH 8.2),12.5 μl DTNB reagent (Ellman's reagent) and 200 μl of methanol. Sampleswere incubated for 5 min at RT, and then extinction measured induplicate for each at 412 nm. Results were compared to a standard curvegenerated with N-acetylcysteine.

Akt Kinase Activity Assay

The effect of sodium selenate on recombinant human Akt1 kinase activitywas determined using the K-LISA™ AKT Activity Kit (Calbiochem #CBA019).This is an ELISA based assay that utilizes a biotinylated peptidesubstrate (GRPRTSSFAEG) that is phosphorylated on the second serine byAkt1, Akt2, Akt3, SGK (Serum Glucocorticoid Kinase), and MSK1.Biotinylated Akt substrate and Akt sample are incubated in the presenceof ATP in wells of Streptavidin-coated 96-well play, which allows forphosphorylation and substrate capture in a single step. Thephosphorylated substrate is detected with a phospho-serine detectionantibody, followed by the HRP-antibody conjugate, and colour developmentwith TMB substrate.

At the start of each assay run, a new aliquot of biotinylated Aktsubstrate working solution was prepared by diluting the stock solution1:100 with dH₂O. The following was then added to each well in thisorder: 10 μl of 5× Kinase Assay Buffer; 10 μl of Biotinylated AktSubstrate working solution; 250 ng of recombinant human Akt1 in 10 μl ofdH₂O; 500 μM sodium selenate or 1 μM staurosporine in 10 μl of dH₂O, orjust dH₂O for positive controls; 10 μl of 5×ATP/MgCl₂ mix to a total of50 μl per well. The plate was sealed with plate-sealer, mixed briefly ona microplate shaker, and incubated for 30 m at 30° C. The kinasereaction was then stopped by adding 10 μl of kinase stop solution toeach well. The contents of each well was discarded, and then washed 3times with 1×ELISA wash solution (prepared by diluting the stock ELISAwas solution 1:20 with dH₂O), inverted and tapped on blotting paperuntil dry. 100 μl of phospho-serine detection antibody working solution(prepared freshly for each assay run by diluting the phospho-serineantibody stock 1:1000 with dH₂O) was then added to each well andincubated for 1 h at RT. The plate was then washed as described above.100 μl of HRP-antibody conjugate working solution (prepared freshly foreach assay run by diluting the HRP-antibody stock 1:1000 with dH₂O) wasthen added to each well at incubated for 1 h at RT. The plate was thenwashed again as described above. 100 μl of TMB substrate was then addedto each well, and the colour allowed to develop for 20 min at RT. Thereaction was halted by adding 100 μl of ELISA stop solution to eachwell, and absorbance read at 450 nm with reference to 590 nm using amicroplate reader.

Intracellular Redox State Assay

Intracellular redox state was determined using2′,7′-dichlorodihydrofluorescein diacetate (DCFDA). DCFDA isnon-fluorescent and freely cell permeable, but in the presence ofreactive oxygen species (ROS) is rapidly converted to the cellimpermeable but highly fluorescent 2′,7′-dichlorofluorescein (DCF).Cells were equilibrated with 5 μM of DCFDA for 15 min prior to varioustreatments. Adherent cells were then harvested, washed twice with PBSand the proportion of fluorescent cells determined immediately by flowcytometry.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims.

Example 10 Effects of Selenate on Tau Phosphorylation Activities Both onMouse Brain Tissues and Human Neuroblastoma Cell Lines

Materials and Methods

Cell Culture.

Human neuroblastoma SY5Y and BE2M17 cell lines were obtained fromJanetta Culvenor (Department of Pathology, University of Melbourne,VIC). SY5Y cells were routinely cultured in RPMI 1640 medium(Invitrogen, Auckland, New Zealand) supplemented with 10% fetal bovineserum (FBS, Invitrogen, GIBCO), 1% non-essential amino acids (Sigma, StLouis, Mo., USA), 10 mM HEPES (Invitrogen, GIBCO), 1 mM sodium pyruvate(Invitrogen, GIBCO) and 1% antibiotic/antimycotic mixture (Invitrogen,GIBCO). BE2M17 cells were cultured in OPTI-MEM I reduced serum medium(Invitrogen, GIBCO) supplemented with 10% FBS (Invitrogen, GIBCO), 1%non-essential amino acids (Sigma, St Louis, Mo., USA), 1 mM sodiumpyruvate (Invitrogen, GIBCO), and 1% antibiotic/antimycotic mixture(Invitrogen, GIBCO). Cells were cultured at 37° C. in 5% CO₂. Sodiumselenate was obtained from Sigma (St Louis, Mo., USA).

Antibodies:

The following antibodies were obtained from Pierce Endogen (Rockford,USA) unless otherwise stated: anti-human PHF-Tau monoclonal (cloneAT100; AT180; AT270), and anti-human Tau monoclonal (clone HT-7).Polyclonal goat anti-mouse immunoglobulins/HRP (Dako, Denmark) andAnti-tubulin (G712A, Promega).

In Vitro:

2×10⁵ SY5Y or BE2M17 cells were plated to each well in a Falcon 6-wellplate, either over coated or uncoated surfaces, and were allowed toattached overnight at 5% CO₂/37° C. in complete growth medium (asdescribed above). The medium was replaced with fresh complete growthmedium containing 100 μM concentration of sodium selenate and werecultured for 3 hours. Cells were cultured over Falcon 6-well plates withsurfaces coated either with 0.1% gelatin (Sigma, St Louis, Mo., USA),matrigel (cat #354234, BD, NSW, Australia) or 0.5 μg/ml fibronectin(Sigma, St Louis, Mo., USA).

In Vivo:

14 weeks old Balb/C Nu Nu male mice were obtained from ARC. Mice receivea single subcutaneous injection of 300 μg sodium selenate per 200 μl inPBS. Mice were sacrificed at various time points following injection andblood and brain tissues specimen were collected.

Lysates Preparation and Immunoblotting:

Cells were washed twice in cold PBS and were lysed in 150 μl of coldRIPA buffer (containing 10% glycerol, 20 mM Tris, 137 mM NaCl, 0.1% SDS,0.05% IGEPAL, 1% Triton X-100, 2 mM EDTA, 10% NaV, 2% NaF and 4×protease inhibitor), similarly, brain tissues were homogenised in dryice using mortar and pestle, and lysed in 150 μl RIPA buffer. Sampleswere incubated on ice for 30 minutes, followed by centrifugation at13000 rpm/10 minutes/4° C. Supernatant was collected and proteinestimation was calculated for each sample by using BCA reagent (Sigma).Equal amounts of protein were loaded into each lane of a 10%SDS-polyacrylamide gel. The proteins were transferred onto PVDF(Millipore) membranes and were blocked with 5% skim milk blotto for 2hours. After blocking the membrane, primary antibodies: eitheranti-human PHF-Tau (AT100 at 1:200, AT180 at 1:1000, and AT270 at1:2500) or anti-human Tau (HT-7 at 1:1000) were incubated overnight at4° C. in 5% skim milk blotto. Prior to secondary antibody incubation,membranes were rinsed 3× followed by 3×5 minutes washed in Tris-BufferedSaline with 0.01% Tween at room temperature. Followed by 1 hourincubation of secondary antibody polyclonal goat anti-mouse (Dako,Denmark) coupled to horse radish peroxidase (HRP) at 1:10000 dilutionsin 5% skim milk blotto/room temperature. Membranes were washed asdescribed above and were detected by using Amersham ECL western blottingdetection reagent (Amersham Bioscience, Buckinghamshire, UK). Membraneswere stripped using 2% SDS/β-Mercaptoethanol stripping buffer andreprobed for tubulin (Promega) to confirm protein loading.

Immunoblotting of human neuroblastoma BE2M17 cell line with anti-humanPHF-Tau in the presence (+) or absence (−) of sodium selenate, undervarious coating surfaces is shown in FIG. 11. Sodium selenate at 100 μMwas prepared in BE2M17 complete growth medium. Cells were cultured withsodium selenate for 3 hours on fibronectin, uncoated (plastic) ormatrigel (as described in methods section).

Results:

Anti-human PHF-Tau clone AT100 was detected at 68 kDa. In the presenceof sodium selenate, PHF-Tau signal appeared to be reduced from cellscultured on fibronectin, and matrigel, in comparison to non-selenatetreated cells.

Anti-human PHF-Tau clone AT180 was detected both at 71 and 67 kDa.Selenate treated cells cultured on fibronectin, plastic (uncoated) andmatrigel appeared to have a weaker PHF-Tau signal compared tonon-selenate treated cells. Cells cultured on matrigel give a strongersignal of PHF-Tau at 67 kDa compared to cells cultured on fibronectinand plastic.

Anti-human PHF-Tau clone AT270 was detected both at 66 and 63 kDa.Selenate treated cells cultured on fibronectin, plastic (uncoated) andmatrigel appeared to have a weaker PHF-Tau signal compared tonon-selenate treated cells. Note, there is only a slight differencebetween selenate treated and non-treated cells cultured on matrigel.

Anti-human Tau clone HT-7 was detected at 68, 64, and 57 kDa. Cellscultured on fibronectin with selenate appeared to reduce Tau expression.

Anti-tubulin was detected showing equal protein loading.

Immunoblotting of human neuroblastoma SY5Y cell lines with anti-humanPHF-Tau in the presence (+) or absence (−) of sodium selenate, undervarious culturing surfaces is shown in FIG. 12. Sodium selenate at 100μM was prepared in SY5Y complete growth medium. Cells were cultured withsodium selenate for 3 hours on gelatin, fibronectin, uncoated (plastic)or matrigel (as described in methods section).

Results:

Anti-human PHF-Tau clone AT270 specifically detected at 70 kDa. Selenatetreated cells cultured on gelatin and fibronectin appeared to have aweaker PHF-Tau signal compared to non-selenate treated cells.

Anti-human Tau clone HT-7 was detected at 60 kDa. Both matrigel andfibronectin appeared to reduced anti-Tau signal. In particularly, cellscultured on fibronectin with selenate appeared to have a weaker signalfor anti-Tau than those cultured without selenate.

Anti-tubulin was detected showing relative equal protein loading.

Immunoblotting of total brain lysates from 14 weeks old Balb/C Nu Numale mice with anti-human PHF-Tau (as indicated) either with (+) orwithout (−) sodium selenate is shown in FIG. 13. Mice treated withsodium selenate were given by subcutaneous injection at 1.5 μg/μl. Inthe absence of sodium selenate (−) mice were subcutaneously injectedwith PBS. Brain tissue was collected at various time points: 0, 2, and 6hours (as described in methods section).

Results:

PBS appeared to have an effect on anti-human PHF Tau signal for allclones studied (AT100, AT180 and AT270). To confirm equal proteinloading all membranes were stripped and reprobed for tubulin asdescribed in the methods section.

FIG. 13A shows the immunoreactivity of anti-human PHF Tau clone AT100 at60 kDa. In the presence of sodium selenate, PHF Tau signal appeared tobe reduced at 2 and 6 hours time point compared to selenate non-treatedcells.

FIG. 13B shows immunoreactivity of anti-human PHF Tau clone AT180 at 60kDa. In the presence of sodium selenate, PHF Tau signal clearly reducedat 2 and 6 hours compared to selenate non-treated cells.

FIG. 13C shows similar immunoreactivity signals of AT270 compared toAT180 clones. It appeared that AT270 clones detected three isoforms ofPHF Tau at 73, 70 and 60 kDa.

FIG. 13D shows the expression of Tau protein in mice brain total lysateseither with (+) or without (−) sodium selenate. Anti-human Tau,specifically clone HT-7 in mice brain tissues appeared to benon-specific.

Example 11 Effects of Sodium Selenate on Behaviour and Tau BrainPathology of Transgenic Mice Over-Expressing Human Tau 441 (TMHT)

Methods

Introduction

The study was designed to evaluate the effects of a treatment withsodium selenate on behaviour and brain morphology of TAU441 transgenic(Tg) TMHT mice (C57BL6 background) over-expressing the human TAU441 genewith two mutations, V337M and R406W, under the control of a brainspecific murine Thy-1 promoter. Behaviour of all Tg mice was evaluatedafter 1.5 and 3 months after treatment in the Open Field (OF) test, theRota Rod (RR) test and the Nose poke curiosity and activity test, thelatter to evaluate curiosity behaviour. At the end of the treatmentadditionally memory and learning were evaluated in the Morris Water Maze(MWM) task. Untreated mice of the baseline group were also tested in theOF test, the RR test, the Nose poke test and the MWM task. Brain TAUpathology, were determined initially in 3 animals per treatment group.

Animals

Male and female Tg TMHT mice expressing human TAU441 bearing themissense mutations V337M and R406W under the regulatory control of thebrain specific murine Thy-1 promoter were used. Mice were generated andbred at JSW-Research, Graz, Austria. The C57BL/6 background for thesemice result in the mice being known as good learners. This mouse modelresembles human Alzheimer's disease tau-pathology. At treatment startmice had an age of 5 months±2 weeks and this was also the age of thebaseline group.

Animal Identification and Housing

Mice were identified with ear markings. They were housed in individualventilated cages (IVCs) on standardized rodent bedding supplied byABEDD®. Each cage contained a maximum of five mice. Mice were keptaccording to the standard operating procedures based on internationalstandards.

Each cage was identified by a colored card indicating the study number,sex, the individual registration numbers (IRN) of the animals, date ofbirth, as well as the screening date and the treatment group allocation.

The temperature during the study was maintained at approximately 24° C.and the relative humidity was maintained at approximately 40-70%.Animals were housed under a constant light-cycle (12 hours light/dark).

Dried, pelleted standard rodent chow (Altromin®) and normal tap waterwere available to the animals ad libitum.

Treatment

Mice were randomly allocated to the groups A (sodium selenate), B(vehicle) and C (baseline). Treatment group A received sodium selenatevia drinking water for 12 weeks while control mice (B) had access tonormal tap water. For application, 1.2 mg sodium selenate was dissolvedin 100 mL of sterile water, the weight was recorded and the bottle wasplaced in the cage. This was done every Monday, Wednesday and Friday,and the water was weighed on exchange to measure consumption.

Behavioural Testing

Open Field Test

The most standardized general measure of locomotor function isspontaneous activity in the Open Field (OF). For the presentinvestigations, a Plexiglas Open Field (48×48 cm; TSE-Systemc®) wasused. The infrared photo beams were placed at 1.4 cm distance around thebox. To detect rearing (standing on the hind paws) another row of photobeams was mounted 4 cm above the first one. The test session lasted for5 minutes to check the mouse behaviour in the new surroundings as wellas habituation. Thereafter the number of fecal boluses was counted, as ameasure of emotionality. The OF was cleaned with 70% ethanol after everymouse to remove odour traces. Testing was performed under standard roomlighting conditions during the light phase of the circadian cycle.

Rota Rod Test

This test was used to detect possible motor deficits of the TAU Tg mice.Investigations were conducted on an accelerating five-lane-Rota Rod(TSE-Systems®). The mice had to complete a program for maximal 300 sec.They started with a speed of 5 rpm and after 120 sec the rod reached aspeed of 60 rpm. After this run, the latency to fall and the speed ofthe rod at that time were calculated.

Nose Poke Curiosity and Activity Test

The hole-board apparatus offers a simple method of measuring theresponses of a mouse to a novel environment and takes advantage of thecurious nature of mice and their tendency to poke their noses intoholes. The investigation of curiosity behaviour was done in OF boxesequipped with hole-boards (16 holes/board). Head dipping into a holeinterrupted the infrared beams just below the edge of each hole. Numberand duration of the head dippings were calculated for each animal duringa 5-minute period.

Morris Water Maze (MWM)

The Morris Water Maze task was conducted in a black circular pool of adiameter of 100 cm. The pool was filled with tap water at a temperatureof 22±1° C. and the pool was virtually divided into four sectors. Atransparent platform (8 cm diameter) was placed about 0.5 cm beneath thewater surface. During the whole test session, except the pretest, theplatform was located in the southwest quadrant of the pool.

One day before the 4 days lasting training session animals had toperform a so called “pre-test” (two 60 second trials) to ensure that thevisual ability of each animal were normal. Only animals that fulfilledthis task continued to the MWM testing.

In the MWM task each mouse had to perform three trials on fourconsecutive days. A single trial lasted for a maximum of one minute.During this time, the mouse had the chance to find the hidden,diaphanous target. If the animal could not find a “way” out of thewater, the investigator guided to or placed the mouse on the platform.After each trial the mouse was allowed to rest on the platform for 10-15seconds. During this time, the mouse had the possibility to orientate inthe surroundings. Investigations took place under dimmed lightconditions, to prevent the tracking system from negative influences(Kaminski; PCS, Biomedical Research Systems). On the walls surroundingthe pool, posters with black, bold geometric symbols (e.g. a circle anda square) were fixed which the mice could use the symbols as landmarksfor orientation.

One swimming group per trial consisted of five to six mice, so that anintertrial time of about five to ten minutes was ensured.

For the quantification of escape latency (the time in seconds the mouseneeded to find the hidden platform and therefore to escape from thewater), of pathway (the length of the trajectory in meters to reach thetarget) and of the abidance in the goal quadrant a computerized trackingsystem was used. The computer was connected to a camera placed above thecentre of the pool. The camera detected the signal of the light emittingdiode (LED), which was fixed with a little hairgrip on the mouse's tail.

Probe Trial

One hour after the last trial on day 4, mice had to fulfill a so-calledprobe trial. At this time, the platform was removed from the pool andduring the one-minute probe trial, the experimenter counted the numberof crossings over the former target position. Additionally the abidancein this quadrant was measured.

Tissue Preparation and Sampling

At the end of the treatment period, and following all behaviouraltesting, at sacrifice from each animal, blood (plasma and serum), CSFand brain were obtained, processed immediately or stored for furtherexperiments.

For that purpose all mice were sedated by standard inhalation anesthesia(Isofluran, Baxter). Cerebrospinal fluid was obtained by bluntdissection and exposure of the foramen magnum. Upon exposure, a Pasteurpipette was inserted to the approximate depth of 0.3-1 mm into theforamen magnum. CSF was collected by suctioning and capillary actionuntil flow fully ceased. Each sample was immediately frozen and kept at−80° C. until ready for further analysis with ELISA technique.

After CSF sampling, each mouse was placed in dorsal recumbence and a26-gauge needle attached to a 1 mL syringe was inserted into the thoraxthrough the diaphragm to an approximate depth of 2 cm. Light suction wasapplied to the needle and placement in the cardiac (ventricular) chamberof the mouse was confirmed by blood flow to the syringe chamber. Bloodwas aspirated until flow ceased, collected in EDTA vials and stored at−20° C. until later use.

After blood sampling transgenic mice were intracardially perfused with0.9% sodium chloride. Brains were rapidly removed, and the right halfwas immersion fixed for 24 hours in freshly prepared 4% Paraformaldehydeand embedded in paraffin for histological investigations. The lefthemisphere was frozen on dry ice and stored at −80° C. for possiblelater biochemical analysis.

In initially nine (9) brain hemispheres (≧3 per Tg animal group)histological evaluations were performed to qualitative and quantitativeevaluate TAU pathology.

Fifteen (15) coronal consecutive sections (Leica SM 2000R) were cut (5μm thick) at each of 5 different brain layers between Bregma −1.82 and−1.34 mm, which were chosen according to the morphology atlas “The MouseBrain” from Paxinos and Franklin (2^(nd) edition), for Gallyas stainingand for determination TAU pathology with specific antibodies (AT180 andHT7). Tissues of all transgenic animals investigated were handled inexactly the same way to avoid bias due to variation of this procedure.Remaining brain hemispheres or tissue not used are saved and storeduntil the sponsor has decided to how to proceed or until end of thestudy.

Immunohistochemical Determination of TAU Pathology

TAU depositions were determined using the monoclonal TAU-antibodiesAT180 and HT7 (Pierce Endogen®). AT180 recognizes PHF-TAU and tanglelike formations [the epitope of this antibody is the phosphorylatedThr231 residue] and HT7 normal human TAU and PHF-TAU [the epitope ofthis antibody has been mapped on human TAU between residue 159 and 163].

Five (5) μm thick coronal paraffin sections from each of the fivedifferent layers were stained with the above described monoclonal mouseanti-human TAU-antibodies (AT180—1:100; HT7—1:1000) and visualized usinga secondary anti-mouse Cy3 (1:500, Jackson Laboratories®). The detailedstaining protocols are set out below:

AT180 Incubation Protocol for the Determination of Human PHF-TAUDepositions

-   -   1.) Deparaffinize and hydrate tissue sections through Tissue        Clear (Sakura®) and graded alcohol (Merck®) series.    -   2.) Wash for 2 minutes in Aqua bidest (Fresenius-Kabi®)    -   3.) Place tissue sections for antigen unmasking in 10% citrate        buffer (Labvision®) for 15 minutes at 95° C. in a steamer and to        cool down for 15 minutes at room temperature.    -   4.) Wash sections for 2×5 minutes in PBS.    -   5.) Block endogenous peroxidase with 1% hydrogen peroxide        (Linaris®) in methanol (Merck®) for 10 minutes at room        temperature.    -   6.) Wash sections for 2×5 minutes in PBS.    -   7.) Block unspecific bindings with MOM-Blocking Reagent        (Vector®) for 60 minutes at room temperature in a damp chamber.    -   8.) Wash sections for 2×5 minutes in PBS.    -   9.) Block unspecific bindings with MOM-Diluent (Vector®) for 5        minutes at room temperature.    -   10.) Incubate with AT180 (Pierce Endogen®; 1:100 in MOM-Diluent)        for 60 minutes at room temperature in a damp chamber.    -   11.) Wash sections for 3×5 minutes in PBS.    -   12.) Block unspecific bindings with 10% Non-Immune Goat-Normal        Serum (Dako®) for 10 minutes at room temperature in a damp        chamber.    -   13.) Wash sections for 2×5 minutes in PBS.    -   14.) Incubate with Cy 3 Goat Anti-Mouse (Jackson®; 1:500 in        MOM-Diluent) for 60 minutes at room temperature in a damp        chamber.    -   15.) Wash sections for 5 minutes in PBS.    -   16.) Wash sections for 5 minutes in Aqua bidest.    -   17.) Cover sections with Moviol and coverslips.

Chemicals and Reagents:

1% Hydrogen Peroxide in Methanol:

60 mL methanol+2 mL 30% hydrogen peroxide+0.6 mL Triton X-100(Amresco®).

MOM-Blocking Reagent:

2 drops of MOM-Mouse IgG Blocking Reagent (from MOM-Kit (Vector®))+2.5mL PBS.

MOM-Diluent:

10 mL PBS+800 μL Protein Concentrate (from MOM-Kit (Vector®)).

Antibody AT180:

1:100 in MOM-Diluent

Antibody Cy3 Goat Anti-Mouse:

1:500 in MOM-Diluent

HT7 Incubation Protocol for the Determination of Normal Human TAU AndPHF-TAU Depositions

-   -   1.) Deparaffinize and hydrate tissue sections through Tissue        Clear (Sakura®) and graded alcohol (Merck®) series.    -   2.) Wash for 2 minutes in Aqua bidest (Fresenius-Kabi®).    -   3.) Place tissue sections for antigen unmasking in 1% citrate        buffer (Labvision®) for 15 minutes at 95° C. in a steamer and to        cool down for 15 minutes at room temperature.    -   4.) Wash sections for 2×5 minutes in PBS.    -   5.) Block endogenous peroxidase with 1% hydrogen peroxide        (Linaris®) in methanol (Merck®) for 10 minutes at room        temperature.    -   6.) Wash sections for 2×5 minutes in PBS.    -   7.) Block unspecific bindings with MOM-Blocking Reagent        (Vector®) for 60 minutes at room temperature in a damp chamber.    -   8.) Wash sections for 2×5 minutes in PBS.    -   9.) Block unspecific bindings with MOM-Diluent (Vector®) for 5        minutes at room temperature.    -   10.) Incubate with HT7 (Pierce Endogen®; 1:1000 in MOM-Diluent)        for 60 minutes at room temperature in a damp chamber.    -   11.) Wash sections for 3×5 minutes in PBS.    -   12.) Block unspecific bindings with 10% Non-Immune Goat-Normal        Serum (Dako®) for 10 minutes at room temperature in a damp        chamber.    -   13.) Wash sections for 2×5 minutes in PBS.    -   14.) Incubate with Cy 3 Goat Anti-Mouse (Jackson®; 1:500 in        MOM-Diluent) for 60 minutes at room temperature in a damp        chamber.    -   15.) Wash sections for 5 minutes in PBS.    -   16.) Wash sections for 5 minutes in Aqua bidest.    -   17.) Cover sections with Moviol and coverslips.

Chemicals and Reagents:

1% Hydrogen Peroxide in Methanol:

60 mL methanol+2 mL 30% hydrogen peroxide+0.6 mL Triton X-100(Amresco®).

MOM-Blocking Reagent:

2 drops of MOM-Mouse IgG Blocking Reagent (from MOM-Kit (Vector®))+2.5mL PBS.

MOM-Diluent:

10 mL PBS+800 μl Protein Concentrate (from MOM-Kit (Vector®)).

Antibody HT7:

1:1000 in MOM-Diluent

Antibody Cy3 Goat Anti-Mouse:

1:500 in MOM-Diluent

Evaluation

Behaviour

In the Open Field (OF) the horizontal and vertical activity, number offecal boli, number and duration of rearing, hyperactivity and time spentin the centre versus time spent in the perimeter of the open field weremeasured.

In the Nose poke curiosity and activity test the number and duration ofthe head dippings was calculated.

In the Rota Rod test the latency to fall and the speed of the rod atthat time were calculated.

In the Morris water maze trials length of swimming path and escapelatencies were recorded. Swimming speed was calculated as swimming pathdivided by escape latency. In the probe trial, a trial with removedplatform, the number of crossings over the former platform position wasrecorded to the data sheet as well as the time spent in each quadrant ofthe pool.

Neuropathlogy

To determine the extent of TAU immunoreactivity in hippocampus andamygdala specialized image analysis software (Image Pro Plus, version4.5.1.29) was used. Following parameters were evaluated and calculated:

-   -   region area of hippocampus and amygdala in each slice    -   absolute area of immunoreactive positive cells in the specific        brain regions hippocampus and amygdala    -   number of immunoreactive positive area relative to the specific        brain region area of the hippocampus and amygdala

Quantification Procedure:

-   -   a) Contrasting the image for better visualization of slice        morphology without applying the contrast to the image.    -   b) Interactive drawing of the hippocampal outlines and        measurement of the hippocampal area (=region area).    -   c) Interactive drawing of the area of interest (AOI), in which        stained objects are detected over an intensity based threshold        level. To determine the threshold value a line histogram was        interactively drawn close to the AOI in an area without visible        immunoreaction. The mean intensity level from all pixels of the        line histogram plus a constant defined the intensity threshold        level. Objects below a size of 7 μm² were excluded.    -   d) Measurement of the area of each object and the sum of stained        area in the AOI.    -   e) Repetition of a-d) for the amygdala.    -   f) Calculation of the relative TAU immunopositive area (=“sum        area of TAU immunoreactivity/region area*100”).    -   g) Automated data export into an Excel spread sheet, including        the parameters “image title, region area, total TAU area and TAU        area in percentage. A field for remarks was used to record image        quality and exclusion criteria, respectively. Exclusion criteria        were missing parts of the slice, many wrinkles or dominant        flaws.    -   h) Closing the image without saving (to keep raw data).

Statistics

Means, standard deviation or SEMs were calculated for all measuredparameters.

Results

General Observations

In general it can be stated that the treatment with sodium selenate didnot lead to any negative side effects or caused any premature deaths.

Behavioural Results

Open Field

While activity parameters (activity, hyperactivity, rearing behaviour)were uninfluenced by sodium selenate treatment, sodium selenate treatedmice showed disturbed thigmotactic behaviour during the first OFsession. This resulted in a significant higher abidance in the centre ofthe Open Field box, meaning less thigmotaxis, between vehicle and sodiumselenate treated animals in the first turn (see FIG. 14—“thigmotaxis”,T-test for treated animals: p=0.019). At the end of treatment in turn 2thigmotactic behaviour normalized to the level of the vehicle controls.

Baseline animals featured higher defecation rates in the OF test versusboth treated groups investigated one and a half months later (ANOVA:p=0.018, p<0.05 for both treated groups) indicating a higher emotionalreaction to the test procedure.

Rota Rod

Motor performance remained unchanged with sodium selenate treatment.

Nose Poke Curiosity and Activity Test

Curiosity behaviour remained unchanged with sodium selenate treatment.

Morris Water Maze

Results of the Morris Water Maze (MWM) performance of the two differenttreatment groups plus baseline (5 months old animals treatment) areshown in FIG. 15 and Table 4 showing the results of the statisticalanalysis. The MWM task revealed pronounced in some cases (see Table 4)even significant differences between sodium selenate treated animals incomparison to the vehicle group and even in comparison to the threemonths younger baseline group.

TABLE 4 Table of Mann Whitney U-test results for Morris Water Mazeresults: TIME LENGTH sel vs vehicle sel vs vehicle n n n n sel vehiclep-values sel vehicle p-values DAY 1 15 16 0.0020 DAY 1 15 16 0.4945 DAY2 15 16 0.0082 DAY 2 15 16 0.4701 DAY 3 15 16 0.0267 DAY 3 15 16 0.6823DAY 4 15 16 0.1015 DAY 4 15 16 0.5196 sel vs baseline sel vs baseline nn n n sel baseline p-values sel baseline p-values DAY 1 15 15 0.0295 DAY1 15 15 0.6236 DAY 2 15 15 0.6827 DAY 2 15 15 0.8063 DAY 3 15 15 0.3669DAY 3 15 15 1.0000 DAY 4 15 15 0.5125 DAY 4 15 15 0.9349 vehicle vsbaseline n vehicle vs basline ve- n n n hicle baseline p-values vehiclebasline p-values DAY 1 16 15 0.5196 DAY 1 16 15 0.6260 DAY 2 16 150.0655 DAY 2 16 15 0.7701 DAY 3 16 15 0.1195 DAY 3 16 15 0.7112 DAY 4 1615 0.1751 DAY 4 16 15 0.6260

The results clearly show the potential of sodium selenate improvingcognitive functions.

Results of Brain Histology and Immunohistochemistry

General Results

In the quantifications of AT180 and HT7 IHC region areas of thehippocampus and the amygdala were highly constant throughout allinvestigated brains, which excludes negative effects on tissue inimmunohistochemical staining steps (e.g. shrinkage, different cuttingcircumstances), and is furthermore a sign that there was no treatmentinduced atrophy. Measured TAU load data were related to the individualregion size in the slice to be able to cope with minimal differences.

HT7 and AT180

The percentage of relative HT7-TAU area in the hippocampus wassignificantly reduced in animals treated with sodium selenate (1.2 mg)versus vehicle (ANOVA: p<0.05, T-Test of treated groups: p=0.04) anduntreated baseline groups (ANOVA: p<0.05; see FIG. 16A). This effect wasmore pronounced in the Amygdala (see FIG. 16B) in which the sodiumselenate treated mice showed significantly reduced percentage ofrelative HT7-TAU area versus untreated baseline group (ANOVA: p<0.01)and versus vehicle treated animals (ANOVA: p<0.05; T-test for treatedonly: p=0.0018).

Similar to HT7-TAU also the percentage of relative AT180-TAU area in thehippocampus was reduced in animals treated with sodium selenate (1.2 mg)versus vehicle (T-Test of treated groups: p=0.04, see FIG. 17A), howeverat lower level of significance. Again this effect was more pronounced inthe Amygdala (see FIG. 17B) in which the sodium selenate treated miceshowed significantly reduced percentage of relative HT7-TAU area versusvehicle group (ANOVA: p<0.05, T-Test of treated groups: p=0.0045) butnot versus untreated baseline group.

AT180 and HT7 immunoreactive positive neurons in hippocampus andamygdala showed massive TAU depositions in neuronal soma, which wasdensely packed with PHF TAU. Sodium selenate treatment visibly reducedTAU load as well as TAU positive cells in the amygdala and neuronallayer of the hippocampal CA1 region.

SUMMARY OF EFFECTS AND CONCLUSIONS

-   -   Less thigmotaxis one and a half month after treatment start        hints at changes in fear related to amygdaloideic structures in        the sodium selenate treated transgenic mice. After prolonged        sodium selenate treatment mice returned to comparable and normal        thigmotactic behavior of vehicle treated controls in the second        Open Field test run before sacrificing.    -   All motor parameters like RotaRod performance, Open Field        activity, hyperactivity and rearing behavior were uninfluenced        by sodium selenate treatment.    -   The treatment with sodium selenate could improve cognitive        abilities tested in the Morris Water Maze. On day 1, 2 and 3        sodium selenate treated animals could find the platform high        significantly (p≦0.01) faster than animals from the vehicle and        on day 1 also faster than the three months younger animals from        the baseline group.    -   Pronounced effects of a treatment with sodium selenate can be        seen when evaluating HT7 immunopositive TAU deposition and TAU        load in hippocampus and amygdala of TMHT Tg mice.    -   Due to the treatment a significant reduction of HT7 positive TAU        area in hippocampus and amygdala compared to the untreated        baseline group or rather in the amygdala also to the vehicle        (H₂O) treated controls can be seen.    -   Results on HT7-TAU deposition are supported by the AT180        incubation. Here a significant reduction of AT180 positive TAU        after treatment with sodium selenate in the hippocampus and        amygdala compared to the vehicle (H₂O) treated controls can be        seen.

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What is claimed is:
 1. A method for the treatment of a tauopathy in asubject in need thereof, said method comprising administering to thesubject an effective amount of selenate or a pharmaceutically acceptablesalt thereof, with the proviso that the pharmaceutically acceptable saltof selenate is not lithium selenate.
 2. The method according to claim 1wherein the tauopathy is selected from presenile dementia, seniledementia, Frontal Temporal Dementia with Parkinsonism 17, progressivesubnuclear palsy, Pick's disease, primary progressive aphasia,frontotemporal dementia and corticobasal dementia.
 3. The methodaccording to claim 1 wherein the tauopathy is Alzheimer's disease. 4.The method according to claim 1 further comprising the administration ofanother therapy for treatment of a tauopathy or a therapy for relievingthe symptoms of a tauopathy.
 5. A method for the prevention of atauopathy in a subject in need thereof, said method comprisingadministering to the subject an effective amount of selenate or apharmaceutically acceptable salt thereof.
 6. The method according toclaim 5 wherein the tauopathy is selected from presenile dementia,senile dementia, Frontal Temporal Dementia with Parkinsonism-17,progressive subnuclear palsy, Pick's disease, primary progressiveaphasia, frontotemporal dementia and corticobasal dementia.
 7. Themethod according to claim 5 wherein the tauopathy is Frontal TemporalDementia with Parkinsonism-17.
 8. The method according to claim 5wherein the tauopathy is Alzheimer's disease.